Anti-LAM and anti-PIM6/LAM monoclonal antibodies for diagnosis and treatment of Mycobacterium tuberculosis infections

ABSTRACT

The present invention broadly provides different compositions, kits, vectors, and methods including monoclonal antibodies directed to epitopes found within lipoarabinomannan (LAM) and phosphatidyl-myo-inositol mannoside 6 (PIM6) for the diagnosis and treatment of Mycobacterium tuberculosis infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 16/910,621, filed Jun. 24, 2020, which is adivisional application of U.S. patent application Ser. No. 16/076,971,filed Aug. 9, 2018, now U.S. Pat. No. 10,729,771, which is a 35 U.S.C.371 National Stage of PCT/US17/16058, filed Feb. 1, 2017, which claimspriority to U.S. Provisional Application No. 62/293,406 filed Feb. 10,2016, all of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

Compositions, kits, vectors, and methods including antibodies directedto epitopes found within lipoarabinomannan (LAM) lipomannan (LM) andphosphatidyl-myo-inositol mannoside 6 (PIM6) for the diagnosis,prevention and treatment of Mycobacterium tuberculosis infections aredescribed herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 17, 2022, isnamed SequenceListing096747.00444.txt and is 28.3 kilobytes in size.

BACKGROUND

A. Mycobacterium Tuberculosis

Tuberculosis (TB) remains one of the world's deadliest communicablediseases, currently infecting approximately ⅓ of the world's population.According to the WHO Global Tuberculosis Report, 2014: Tuberculosis, in2013, an estimated 9.0 million people developed TB, and 1.5 million diedfrom the disease. Although there currently are effective drugs availablefor TB, these require lengthy treatments with multiple antibiotics, andare increasingly compromised by the development of multi-drug resistant(MDR-TB) strains, which currently are responsible for about 3.5% ofrecent infections. These strains are much harder to treat and havesignificantly poorer cure rates. Also spreading are extensivelydrug-resistant TB (XDR-TB) strains, which are even more expensive anddifficult to treat than MDR-TB strains, and have now been reported in100 countries around the world. Consequently, new approaches are neededfor the earlier diagnosis and treatment of TB infections.

B. Lipoarabinomannan (LAM)

The glycolipid lipoarabinomannan (LAM) is a major structural andantigenic component of the cell wall of members of the Mycobacteriumtuberculosis-complex, and it mediates a number of important functionsthat promote productive infection and disease development. LAM is alsoan important immunodiagnostic target for detecting active infection withTB, especially in patients co-infected with HIV-1, and a potentialvaccine target. Despite the importance of LAM as an immunodiagnostictarget and its significant role in the physiology of M.tb infection andpathogenicity, surprisingly little is known about the nature of thehuman humoral response towards this antigen. Previously availableLAM-specific monoclonal antibodies have been derived from mice immunizedwith LAM purified from either Mycobacterium leprae or Mycobacteriumtuberculosis, and there have been no descriptions of any humanmonoclonal antibodies against LAM that have been induced in responseeither to immunization or to infection by Mycobacterium tuberculosis.

Lipomannan (LM)—is the immediate precursor to LAM and contain aphosphatidyl-myo-inositol domain modified by a mannan domain comprisedof an α(1→6)-linked Manp backbone substituted with shortα(1→2)-mannopyranosyl side chains, but with no arabinose side chains.

C. Phosphatidyl-Myo-Inositol Mannoside 6 (PIM6)

PIM6 is a product of PIM2, a common precursor to LM and LAM. The core ofthese molecules is a myo-inositol structure glycosylated with a Manpunit at positions 2 and 6. In PIM6, the Manp unit at positions 6 isfurther substituted by two terminal α-Manp(1→2)-linked sugars identicalto the mannose cap on ManLAM. These molecules are acylated by as many as4 fatty acid chains, attached to the inositol head group and to the coreMan residue, which non-covalently anchor these molecules to the innerand outer membranes of the cell envelope. PIBM6 was reported to bind toC-type lectins and DC-SIGN, the major receptor on dendritic cells, andto be a strong TLR2 agonist and enhancer of HIV-1 replication thatpossesses potent anti-inflammatory activities.

SUMMARY OF THE INVENTION

Described herein are novel anti-LAM and anti-PIM6/LAM monoclonalantibodies (mAbs) for diagnosis and treatment of Mycobacteriumtuberculosis infections. The isolation and characterization of thesenovel human antibodies specific for glycolipids of Mycobacteriumtuberculosis, including human mAbs specific for LAM epitopes, and ahuman mAb specific for an epitope shared by LAM and PIM6, are describedbelow.

Accordingly, described herein is a human monoclonalanti-lipoarabinomannan (anti-LAM) antibody, or an antigen-bindingportion thereof, that specifically binds to a LAM epitope including anAra4 structure, an Ara6 structure, or a combination thereof, wherein theanti-LAM antibody includes a CDR1 variable light region having at least80% identity with SEQ ID NO: 1 or antigenic fragments thereof, a CDR2variable light region having at least 80% identity with SEQ ID NO: 2 orantigenic fragments thereof, a CDR3 variable light region having atleast 80% identity with SEQ ID NO: 3 or SEQ ID NO: 26 or antigenicfragments thereof, a CDR1 variable heavy region having at least 80%identity with SEQ ID NO: 4 or antigenic fragments thereof, a CDR2variable heavy region having at least 80% identity with SEQ ID NO: 5 orantigenic fragments thereof, and a CDR3 variable heavy region having atleast 80% identity with SEQ ID NO: 6 or SEQ ID NO: 23 or antigenicfragments thereof. The human monoclonal anti-LAM antibody orantigen-binding portion thereof can include a heavy chain variableregion including the amino acid sequences of SEQ ID NO:21 and SEQ IDNO:23, and a light chain variable region including the amino acidsequences of SEQ ID NO: 24 and SEQ ID NO:26. The anti-LAM antibody canbe an scFv-IgG, and IgGa or an IgM antibody. An example of an ant-LAMantibody is A194.

Also described herein is a human monoclonal anti-LAM antibody or anantigen-binding portion thereof, that specifically binds to a LAMepitope including at least one of: a mannose-capped Ara4 structure and amannose-capped Ara6 structure. The anti-LAM antibody can include a CDR1variable light region having at least 80% identity with SEQ ID NO: 7 orantigenic fragments thereof, a CDR2 variable light region having atleast 80% identity with SEQ ID NO: 8 or antigenic fragments thereof, aCDR3 variable light region having at least 80% identity with SEQ ID NO:9 or SEQ ID NO: 32 or antigenic fragments thereof, a CDR1 variable heavyregion having at least 80% identity with SEQ ID NO: 10 or antigenicfragments thereof, a CDR2 variable heavy region having at least 80%identity with SEQ ID NO: 11 or antigenic fragments thereof, and a CDR3variable heavy region having at least 80% identity with SEQ ID NO: 12 orSEQ ID NO: 29 or antigenic fragments thereof. The antibody can include aheavy chain variable region including the amino acid sequence of SEQ IDNO:43 and a light chain variable region including the amino acidsequence of SEQ ID NO:44. The anti-LAM antibody can be, for example, anIgM or IgA antibody. An example of an anti-LAM antibody is P3B09.

Further described herein is a human monoclonal anti-LAM antibody, or anantigen-binding portion thereof, that specifically binds to a LAMepitope including an α-Manp(1→2) linked structure attached at anonreducing end of Ara4 or Ara6, wherein the anti-LAM antibody includesa CDR1 variable light region having at least 80% identity with SEQ IDNO: 7 or antigenic fragments thereof, a CDR2 variable light regionhaving at least 80% identity with SEQ ID NO: 8 or antigenic fragmentsthereof, a CDR3 variable light region having at least 80% identity withSEQ ID NO: 9 or antigenic fragments thereof, a CDR1 variable heavyregion having at least 80% identity with SEQ ID NO: 10 or antigenicfragments thereof, a CDR2 variable heavy region having at least 80%identity with SEQ ID NO: 11 or antigenic fragments thereof, and a CDR3variable heavy region having at least 80% identity with SEQ ID NO: 12 orantigenic fragments thereof. The anti-LAM antibody (e.g., P3B09) can be,for example, an IgM or IgA antibody.

Yet further described herein is a human monoclonal anti-PIM6/LAMantibody, or an antigen-binding portion thereof, that specifically bindsto an epitope present in LAM and PIM6, the epitope including at leastone polymannose structure. The epitope is in the PIM6 mannan domain, andis also present in mycobacterial lipomannan (LM). The anti-PIM6/LAMantibody can include a CDR1 variable light region having at least 80%identity with SEQ ID NO: 13 or antigenic fragments thereof, a CDR2variable light region having at least 80% identity with SEQ ID NO: 14 orantigenic fragments thereof, a CDR3 variable light region having atleast 80% identity with SEQ ID NO: 15 or antigenic fragments thereof, aCDR1 variable heavy region having at least 80% identity with SEQ ID NO:16 or antigenic fragments thereof, a CDR2 variable heavy region havingat least 80% identity with SEQ ID NO: 17 or antigenic fragments thereof,and a CDR3 variable heavy region having at least 80% identity with SEQID NO: 18 or antigenic fragments thereof. The antibody can, for example,include a heavy chain variable region including the amino acid sequenceof SEQ ID NO:47 and a light chain variable region including the aminoacid sequence of SEQ ID NO:48. The anti-PIM6/LAM antibody can be, forexample, an IgM, IgA or IgG antibody. An example of an anti-PIM6/LAMantibody is P95C1.

Also described herein is a kit for detecting at least one LAM epitope.The kit includes (a) at least a first anti-LAM antibody that bindsspecifically to a LAM epitope; (b) a support to which the at least firstanti-LAM antibody is bound; (c) a detection antibody that bindsspecifically to LAM, or specifically to the at least first anti-LAMantibody, wherein the detection antibody is labeled with a reportermolecule; and (d) a buffer. The at least first anti-LAM antibody is, forexample, a human monoclonal anti-LAM antibody as described herein. Thedetection antibody can be, for example, a second anti-LAM antibody thatbinds specifically to LAM. In some embodiments, the at least one of thefirst anti-LAM antibody and the second anti-LAM antibody is an scFv-IgGor IgM antibody and includes a CDR1 variable light region having atleast 80% identity with SEQ ID NO: 1 or antigenic fragments thereof, aCDR2 variable light region having at least 80% identity with SEQ ID NO:2 or antigenic fragments thereof, a CDR3 variable light region having atleast 80% identity with SEQ ID NO: 3 or SEQ ID NO: 26 or antigenicfragments thereof, a CDR1 variable heavy region having at least 80%identity with SEQ ID NO: 4 or antigenic fragments thereof, a CDR2variable heavy region having at least 80% identity with SEQ ID NO: 5 orantigenic fragments thereof, and a CDR3 variable heavy region having atleast 80% identity with SEQ ID NO: 6 or SEQ ID NO:23 or antigenicfragments thereof. In some embodiments of the kit, at least one of thefirst anti-LAM antibody and the second anti-LAM antibody includes aheavy chain variable region including the amino acid sequences of SEQ IDNO:21 and SEQ ID NO:23, and a light chain variable region including theamino acid sequences of SEQ ID NO: 24 and SEQ ID NO:26.

Still further described herein is a method of diagnosing an activetuberculosis infection in an individual including: (a) obtaining asample from an individual that includes or is suspected of includingLAM; (b) treating said sample to expose individual LAM epitopes; (c)contacting said sample with at least a first antibody that bindsspecifically to a first epitope on said LAM; (d) contacting said samplewith a detection antibody that binds specifically to LAM, orspecifically to the at least first antibody; (e) detecting binding ofthe at least first antibody to said first epitope on LAM; and (f)diagnosing said patient as having an active tuberculosis infection, thebinding of the at least first antibody to said first epitope on LAMindicating an active tuberculosis infection. The at least first antibodyis, for example, a human monoclonal anti-LAM antibody or humanmonoclonal anti-PIM6/LAM antibody as described herein. The detectionantibody can be, for example, an anti-LAM antibody that bindsspecifically to LAM. In some embodiments of the method, the at leastfirst antibody and the detection antibody each include a CDR1 variablelight region having at least 80% identity with SEQ ID NO: 1 or antigenicfragments thereof, a CDR2 variable light region having at least 80%identity with SEQ ID NO: 2 or antigenic fragments thereof, a CDR3variable light region having at least 80% identity with SEQ ID NO: 3 orSEQ ID NO: 26 or antigenic fragments thereof, a CDR1 variable heavyregion having at least 80% identity with SEQ ID NO: 4 or antigenicfragments thereof, a CDR2 variable heavy region having at least 80%identity with SEQ ID NO: 5 or antigenic fragments thereof, and a CDR3variable heavy region having at least 80% identity with SEQ ID NO: 6 orSEQ ID NO: 23 or antigenic fragments thereof. In some embodiments of themethod, at least one of the first antibody and the detection antibody isan scFv-IgG or IgM antibody and includes a CDR1 region having a variablelight region having at least 80% identity with SEQ ID NO: 1 or antigenicfragments thereof, a CDR2 variable light region having at least 80%identity with SEQ ID NO: 2 or antigenic fragments thereof, a CDR3variable light region having at least 80% identity with SEQ ID NO: 3 orSEQ ID NO: 26 or antigenic fragments thereof, a CDR1 variable heavyregion having at least 80% identity with SEQ ID NO: 4 or antigenicfragments thereof, a CDR2 variable heavy region having at least 80%identity with SEQ ID NO: 5 or antigenic fragments thereof, and a CDR3variable heavy region having at least 80% identity with SEQ ID NO: 6 orSEQ ID NO: 23 or antigenic fragments thereof. In some embodiments, theindividual is a human.

Also described herein is a method of treating a tuberculosis infectionin an individual (e.g., a human). The method includes administering tosaid individual a therapeutically effective amount of at least one humanmonoclonal anti-LAM antibody or human monoclonal anti-PIM6/LAM antibodyas described herein. The method can further include administering tosaid individual a therapeutically effective amount of at least oneantibiotic. The tuberculosis infection can be a multi-drug resistant(MDR-TB) tuberculosis infection.

Further described herein are nucleotide sequences encoding the heavychains and light chains (including variable regions) of the antibodiesdescribed herein.

A. Anti-LAM Antibodies and Anti-PIM6/LAM Antibodies

In some embodiments, the invention provides an anti-LAM antibody, or anantigen binding portion thereof. In some embodiments, the inventionprovides an anti-PIM6/LAM antibody, or an antigen binding portionthereof. An anti-LAM antibody (or antigen binding portion thereof) asdescribed herein binds specifically to a LAM epitope. An anti-PIM6/LAMantibody (or antigen binding portion thereof) as described herein bindsspecifically to both a LAM epitope and a PIM6 epitope. In someembodiments, the LAM and PIM6 epitopes are derived from variousmycobacterial species. In further embodiments, the various mycobacterialspecies are virulent members of the Mycobacterium tuberculosis-complex.In yet further embodiments, the mycobacterial species is Mycobacteriumtuberculosis. In some embodiments, the anti-LAM antibody oranti-PIM6/LAM antibody is a monoclonal antibody (mAb). In furtherembodiments, the anti-LAM antibody or anti-PIM6/LAM antibody is a humanmonoclonal anti-LAM antibody or human monoclonal anti-PIM6/LAM antibody,respectively. In other embodiments, the anti-LAM antibody oranti-PIM6/LAM antibody is a humanized monoclonal anti-LAM antibody oranti-PIM6/LAM antibody, respectively. In some embodiments, the anti-LAMantibody binds to Ara4 and Ara6 structures.

In some embodiments, the LAM epitope is an uncapped arabinose chain. Insome embodiments the LAM epitope is an uncapped or single mannose cappedarabinose chain, with or without a terminal MTX substitution.

In some embodiments, the LAM epitope is a mannose-capped Ara4 structureand a mannose-capped Ara6 structure. In other embodiments, the anti-LAMantibody specifically binds to an α(1□2)-linked dimannose structure,which may be joined either to an Ara4/Ara6 structure, or to apolymannose structure (FIG. 8 ). In some embodiments, the PIM6 epitopeincludes at least one polymannose structure also present inmycobacterial lipomannan (LM). In some embodiments the anti-PIM6/LAMantibody specifically binds to a PIM6 epitope that includes at least onepolymannose structure in the PIM6 mannan domain. In some embodiments,the LAM epitope includes at least one methylthioxylose (MTX) ormethylsylfinylxylofuranosyl (MSX) substitution. In some embodiments, theLAM epitope includes at least one phosphatidyl-myo-inositol substitution(PILAM). In some embodiments, the LAM epitope is an arabinose chaincapped with at least one mannose, i.e. mannosylated Man-LAM epitope. Infurther embodiments, the capped arabinose chain includes Ara4 and/orAra6 structures. In some embodiments, the Man-LAM epitope includesmono-mannose substituted arabinose chains, di-mannose substitutedarabinose chains, tri-mannose substituted arabinose chains, orcombinations thereof. In some embodiments, the Man-LAM epitope includesdi-mannose or tri-mannose capped Ara4 and/or Ara6 structures. In someembodiments, the Man-LAM epitope is di-mannose capped Ara6. In someembodiments, the anti-LAM antibody or anti-PIM6/LAM antibody includes anIgG antibody. In further embodiments, the IgG anti-LAM antibody oranti-PIM6/LAM antibody includes a subclass of IgG1, IgG2 or IgG3. Insome embodiments, the anti-LAM antibody or anti-PIM6/LAM antibody is notan IgG antibody. In other embodiments, the anti-LAM antibody oranti-PIM6/LAM antibody includes an IgA antibody. In other embodiments,the anti-LAM antibody or anti-PIM6/LAM antibody includes an IgMantibody. In some embodiments, the anti-LAM antibody or anti-PIM6/LAMantibody includes a second isotype that has been switched from theisotype originally isolated. In some embodiments, the anti-LAM antibodyor anti-PIM6/LAM antibody includes a recombinant antibody. In someembodiments, the recombinant antibody includes a multivalent IgMantibody. In further embodiments, the recombinant antibody includes apentavalent IgM antibody. In other embodiments, the recombinant antibodyincludes an ScFv-IgG antibody, in which a single chain Fv fragment ofone antibody is joined to the N-terminus of the heavy chain of that or adifferent anti-LAM mAb. In further embodiments, the recombinant antibodyincludes a multivalent ScFv-IgG antibody. In further embodiments, therecombinant antibody includes a homologous tetravalent ScFv-IgGantibody, in which the scFv domains were derived from the variableregions of the IgG present in the construct. In yet further embodiments,the recombinant antibody includes a heterologous tetrameric scFv-IgGantibody in which the scFv regions were derived from a differentanti-LAM antibody or anti-PIM6/LAM antibody as the IgG region included.In some embodiments, the scFv domain includes a leader sequence joinedto the variable heavy (VH) region of second anti-LAM antibody oranti-PIM6/LAM antibody which is joined to the variable light (VL) domainof said anti-LAM antibody or anti-PIM6/LAM antibody. In otherembodiments, the scFv domain includes a leader sequence joined to thevariable light chain region of a first anti-LAM antibody oranti-PIM6/LAM antibody which is joined to the variable heavy (VH) regionof a second anti-LAM antibody or anti-PIM6/LAM antibody. In someembodiments, the anti-LAM antibody is an isolated anti-LAM antibody thatspecifically binds to a LAM epitope (e.g., one of Ara4 and Ara6 orcombinations thereof, an α(1→2)-linked dimannose structure, which may bejoined either to an Ara4/Ara6 structure, or to a polymannose structure).In some embodiments, the anti-LAM antibody does not compete with CS-35and FIND25. In some embodiments, the anti-PIM6/LAM antibody is anisolated anti-PIM6/LAM antibody that specifically binds to at least onepolymannose structure in mycobacterial lipomannan (LM).

In some embodiments, the anti-LAM antibody or anti-PIM6/LAM antibodyincludes a flexible linker. In some embodiments, the flexible linkerjoins the corresponding heavy and light chain domains into a singlechain molecule. In some embodiments, the flexible linker connects animmunoglobulin light chain (IgVL) to an immunoglobulin heavy chain(IgVH). In further embodiments, the flexible linker is comprised of theformula (GGSGG)n (SEQ ID NO:19), wherein n is any positive integerbetween 1 and 200 and any ranges in between, e.g. 1 to 5, 1 to 10, 1 to15, 1 to 25, 1 to 50, 5 to 10, 5 to 25, 10 to 25, 10 to 50, 1 to 100, 1to 150, and all intervening ranges.

In some embodiments, the anti-LAM antibody (e.g., P30B9, A194-01) has atleast one (e.g., one, two, three) complementarity determining region(CDR) (e.g. CDR1, CDR2, CDR3). In some embodiments, the variable lightregion of CDR1 consists essentially of SEQ ID NO: 1 or antigenicfragments thereof. In other embodiments, the variable light region ofCDR1 region consists essentially of SEQ ID NO: 7 or antigenic fragmentsthereof. In other embodiments, the variable light region of CDR1 regionconsists essentially of SEQ ID NO: 13 or antigenic fragments thereof. Insome embodiments, the variable heavy region of CDR1 consists essentiallyof SEQ ID NO: 4 or antigenic fragments thereof. In other embodiments,the variable heavy region of CDR1 region consists essentially of SEQ IDNO: 10 or antigenic fragments thereof. In other embodiments, thevariable heavy region of CDR1 region consists essentially of SEQ ID NO:16 or antigenic fragments thereof.

In some embodiments, the variable light region of CDR2 consistsessentially of SEQ ID NO: 2 or antigenic fragments thereof. In otherembodiments, the variable light region of CDR2 consists essentially ofSEQ ID NO: 8 or antigenic fragments thereof. In other embodiments, thevariable light region of CDR2 consists essentially of SEQ ID NO: 14 orantigenic fragments thereof. In some embodiments, the variable heavyregion of CDR2 consists essentially of SEQ ID NO: 5 or antigenicfragments thereof. In other embodiments, the variable heavy region ofCDR2 region consists essentially of SEQ ID NO: 11 or antigenic fragmentsthereof. In other embodiments, the variable heavy region of CDR2 regionconsists essentially of SEQ ID NO: 17 or antigenic fragments thereof.

In some embodiments, the variable light region of CDR3 consistsessentially of SEQ ID NO: 3 or antigenic fragments thereof. In otherembodiments, the variable light region of CDR3 consists essentially ofSEQ ID NO: 9 or antigenic fragments thereof. In other embodiments, thevariable light region of CDR3 consists essentially of SEQ ID NO: 15 orantigenic fragments thereof. In some embodiments, the variable heavyregion of CDR3 consists essentially of SEQ ID NO: 6 or antigenicfragments thereof. In other embodiments, the variable heavy region ofCDR3 region consists essentially of SEQ ID NO: 12 or antigenic fragmentsthereof. In other embodiments, the variable heavy region of CDR3 regionconsists essentially of SEQ ID NO: 18 or antigenic fragments thereof.

In some embodiments, the anti-PIM6/LAM antibody (e.g., P95C1) has atleast one (e.g., one, two, three) CDR (e.g., CDR1, CDR2, CDR3). In someembodiments, the variable light region of CDR1 consists essentially ofSEQ ID NO: 13 or antigenic fragments thereof. In some embodiments, thevariable heavy region of CDR1 consists essentially of SEQ ID NO: 16 orantigenic fragments thereof. In some embodiments, the variable lightregion of CDR2 consists essentially of SEQ ID NO: 14 or antigenicfragments thereof. In some embodiments, the variable heavy region ofCDR2 consists essentially of SEQ ID NO: 17 or antigenic fragmentsthereof. In some embodiments, the variable light region of CDR3 consistsessentially of SEQ ID NO: 15 or antigenic fragments thereof. In someembodiments, the variable heavy region of CDR3 consists essentially ofSEQ ID NO: 18 or antigenic fragments thereof.

B. Diagnostic Kits and Methods

In some embodiments, the present invention provides kits for detectingthe presence of LAM and/or PIM6 in biological fluids of a human subject.In some embodiments the components of this assays are assembled in alateral flow device (see World Health Organization 2015, The use oflateral flow urine lipoarabinomannan assay (LF-LAM) for the diagnosisand screening of active tuberculosis in people living with HIV). In someembodiments, the kits include a first anti-LAM (e.g., A194-01, P30B9) oranti-PIM6/LAM (e.g., P95C1) capture antibody, a second anti-LAM oranti-PIM6/LAM detector (detection) antibody labeled with a reportermolecule, a support for which the first anti-LAM or anti-PIM6/LAMantibody is bound to, and a buffer. In some embodiments, at least one ofthe first anti-LAM or anti-PIM6/LAM antibody and the second anti-LAM oranti-PIM6/LAM antibody is a human monoclonal anti-LAM antibody thatbinds specifically to one of Ara4 and Ara6 or combinations thereof, or ahuman monoclonal anti-PIM6/LAM antibody that binds specifically to themannan domain of LAM (and lipomannan (LM)). In some embodiments, thefirst anti-LAM antibody and the second anti-LAM antibody bind to thesame LAM epitopes which are present in multiple copies on a single LAMmolecule. In other embodiments, the first anti-LAM antibody and thesecond anti-LAM antibody bind to different epitopes present on a singleLAM molecule. The LAM and PIM6 epitopes may be any of the LAM and PIM6epitopes described herein. In other embodiments, a third detector(detection) antibody is included which binds to a non-competing site ofthe second antibody. In some embodiments, the first antibody and thesecond antibody are of the same isotype. In other embodiments, the firstantibody and the second antibody are different isotypes. In someembodiments of a capture assay, only either the capture antibody or thedetection antibody is an anti-LAM antibody (e.g., A194-01, P30B9) or ananti-PIM6/LAM antibody (e.g., P95C1) as described herein.

The antibodies described herein can be used for additional detection anddiagnostic applications. For example, in one diagnostic assay, one ormore of the antibodies described herein (e.g., A194-01, P30B9, P95C1)can be used to stain tissues obtained from patients to detect thepresence of LAM in lesions suspected of containing TB or TB-infectedcells (e.g., granulomas). This can be done, for example, with a singleantibody as described herein (e.g., A194-01, P30B9, P95C1) that isconjugated with a label that allows sensitive detection. In such amethod or assay, detection by P95C1 of PIM6 or related molecules can beachieved in infected tissues. In another example, P95C1 can be used in aPIM6 competition assay, in which the capture of a labeled form of PIM6by immobilized P95C1 is competed by soluble PIM6 present in a biologicalfluid (e.g., blood or urine) of a suspect. In the absence of solublePIM6, this would result in the capture of a signal, which would becompeted by the presence of soluble PIM6 (see World Health Organization,2015 Policy Guidance—The use of lateral flow urine lipoarabinomannanassay (LF-LAM) for the diagnosis and screening of active tuberculosis inpeople living with HIV).

In some embodiments, the present invention provides kits fordistinguishing between a pathogenic member of the Mycobacteriumtuberculosis-complex and a nonpathogenic member of the Mycobacteriumtuberculosis-complex. In some embodiments, the anti-LAM antibody is ahuman monoclonal anti-LAM antibody that binds specifically to one ofAra4 and Ara6 structure with or without a Man or MTX-Man substitution orcombinations thereof, or anti-PIM6/LAM antibody that specifically bindsat least one polymannose structure in PIM6 or in the LAM mannan domain.In some embodiments, the anti-LAM antibody specifically binds to aMan-LAM epitope including di-mannose substituted side chains,tri-mannose substituted side chains, or combinations thereon. In furtherembodiments, the anti-LAM antibody specifically binds to Man-LAMepitopes includes di-mannose or tri-mannose capped Ara4 and/or Ara6structures. In yet further embodiments, the anti-LAM antibodyspecifically binds to di-mannose capped Ara6 structures.

In some embodiments, the present invention provides methods fordiagnosing an active tuberculosis infection in an individual. In someembodiments the anti-LAM or anti PIM6/LAM antibody can be modified witha sensitive tag and used to identify mycobacterial PIM6 or LAM-relatedmaterial in a tissue sample, as a diagnostic for TB infection andlocalization. In some embodiments, the method involves the capture ofsoluble LAM, and includes the steps of (a) obtaining a sample from anindividual that includes LAM; (b) treating the sample to isolate orexpose said LAM, (c) capturing said isolated or exposed LAM with a firstanti-LAM antibody that binds to a first epitope on said LAM; (d)contacting said isolated or exposed LAM with a second anti-LAM antibody,wherein said second anti-LAM antibody binds to a second epitope on saidLAM; (e) detecting the binding of at least one of said first anti-LAMantibody and said second anti-LAM antibody to said LAM; and (f)diagnosing said patient as having an active tuberculosis infection,wherein said presence of binding of at least one of said first anti-LAMantibody and said second anti-LAM antibody to said LAM indicates anactive tuberculosis infection. In some embodiments, at least one of thefirst anti-LAM antibody and the second anti-LAM antibody is a humanmonoclonal anti-LAM antibody that binds specifically to one of Ara4 andAra6 or combinations thereof. In some embodiments, at least one of thefirst and second antibodies is a human monoclonal anti-PIM6/LAM antibodythat specifically binds to at least one polymannose structure in the LAMmannan domain. In further embodiments, the first antibody and the secondantibody are different isotypes. In some embodiments, at least one ofthe first antibody and the second antibody are recombinant antibodies.In other embodiments, neither the first antibody nor the second antibodyare recombinant antibodies. In yet other embodiments, both the firstantibody and the second antibody are recombinant antibodies.

In some embodiments, the present invention provides methods ofquantifying the amount of LAM and/or PIM6 present in a sample. In someembodiments, the method includes the steps of (a) obtaining a samplethat includes LAM and/or PIM6; (b) contacting said sample with ananti-LAM antibody and/or an anti-PIM6 antibody; (c) detecting thepresence of specific binding of the anti-LAM antibody to said LAM and/orthe binding of the anti-PIM6/LAM antibody to said LAM or said PIM6; and(d) quantifying the amount of LAM or PIM6 in said sample. In someembodiments, the anti-LAM antibody is a human monoclonal anti-LAMantibody that binds specifically to one of Ara4 and Ara6 or combinationsthereof. In some embodiments, the anti-PIM6/LAM antibody is a humanmonoclonal anti-PIM6/LAM antibody that binds specifically to at leastone polymannose structure in the PIM6 mannan domain (e.g., to at leastone polymannose structure in mycobacterial lipomannan (LM)). In someembodiments, quantifying said amount of LAM and/or PIM6 is achieved bycomparing the signal intensity to that of a serially diluted controlsample having a known concentration of LAM and/or PIM6.

In some embodiments the present invention provides methods fordiagnosing an individual as being infected with Mycobacteriumtuberculosis. In some embodiments, the method includes the steps of (a)obtaining a sample that includes LAM or PIM6; (b) contacting said samplewith an anti-LAM antibody and/or an anti-PIM6 antibody, wherein theanti-LAM antibody binds specifically to a LAM epitope including Man-LAMhaving at least one at least one 5-deoxy-5-methylthiopentofuranosyl(MTX) substitution, and the anti-PIM6/LAM antibody binds specifically toan epitope including at least one polymannose structure in the LAMmannan domain, and (c) detecting the presence of specific binding of theanti-LAM antibody to said Man-LAM and/or the presence of specificbinding of the anti-PIM6/LAM antibody to said PIM6. In some embodiments,the anti-LAM antibody is a human monoclonal anti-LAM antibody that bindsspecifically to one of Ara4 and Ara6 or combinations thereof. In someembodiments, the anti-PIM6/LAM antibody is a human monoclonalanti-PIM6/LAM antibody (e.g., P95C1) that binds specifically to at leastone polymannose structure in the PIM6 mannan domain.

In some embodiments the method includes an amplification step thatincreases the sensitivity of the detection method. Examples involve thegeneration of additional target sites by the use of Tyramide SignalAmplification kit (Perkin-Elmer) or the amplification of the initialsignal by the use of the ELISA Amplification System (Thermo Fisher).

In some embodiments, the present invention provides methods ofdifferentiating between a pathogenic member of the Mycobacteriumtuberculosis-complex and a nonpathogenic member of the Mycobacteriumtuberculosis-complex. In some embodiments, the method includes the stepsof (a) obtaining a sample that comprises LAM and/or PIM6; (b) contactingsaid sample with an anti-LAM antibody that binds specifically to aMan-LAM epitope that includes di-mannose substituted side chains,tri-mannose substituted side chains, or combinations thereof, or with ananti-PIM6/LAM antibody that binds specifically to at least onepolymannose structure in the PIM6 mannan domain; and (c) detecting thepresence of specific binding of the anti-LAM antibody to said Man-LAM,or the presence of the specific binding of the anti-PIM6/LAM antibody tosaid at least one polymannose structure in the PIM6 mannan domain,wherein the presence of said specific binding indicates the presence ofa pathogenic member of the Mycobacterium tuberculosis-complex. In someembodiments, the anti-LAM antibody is a human monoclonal anti-LAMantibody that binds specifically to one of Ara4 and Ara6 or combinationsthereof. In further embodiments, the Man-LAM epitope includes di-mannoseor tri-mannose capped Ara4 and/or Ara6 structures. In yet furtherembodiments, the Man-LAM epitope is di-mannose capped Ara6. In someembodiments, the anti-PIM6/LAM antibody is a human monoclonalanti-PIM6/LAM antibody that binds specifically to at least onepolymannose structure in the PIM6 mannan domain.

C. Therapeutic Compositions, Methods, Vaccines, and Vectors

In some embodiments, the present invention provides methods for treatinginfection by a virulent member of the Mycobacterium tuberculosis-complexin an individual. In some embodiments, the method includes administeringa therapeutically effective amount of at least one anti-LAM antibody oranti-PIM6/LAM antibody to an individual exposed to infectious M.tb. Infurther embodiments, the method includes administration of at least oneantibiotic. In some embodiments, the TB infection is active. In otherembodiments, the TB infection is latent. In some embodiments, theinfection is with a multiple-drug resistant (MDR) strain oftuberculosis. In other embodiments, the infection is with anextensively-drug resistant (XDR) strain of tuberculosis.

In some embodiments, the present invention provides a combinationtherapy for treating infection by a virulent member of the Mycobacteriumtuberculosis-complex in an individual. In some embodiments, the methodincludes administering a therapeutically effective amount of a firstanti-LAM antibody that specifically binds to a first LAM epitopeincluding at least one of unsubstituted LAM, mono-mannonsylated Man-LAM,MSX-substituted LAM, and combinations thereof or a first anti-PIM6/LAMantibody that specifically binds to at least one polymannose structurein the PIM6 and LAM mannan domain; and administering a therapeuticallyeffective amount of a second anti-LAM antibody that specifically bindsto a second LAM epitope including at least one of di-mannose substitutedMan-LAM, tri-mannose substituted Man-LAM, and combinations thereof. Insome embodiments, the first antibody and the second antibody areadministered simultaneously (e.g., in a single composition, or in twocompositions administered at the same time). In other embodiments, thefirst antibody and the second antibody are administered at differenttime points. In some embodiments, at least one of the first anti-LAMantibody and the second anti-LAM antibody is a human monoclonal anti-LAMantibody that binds specifically to one of Ara4 and Ara6 or combinationsthereof. In some embodiments, the anti-PIM6/LAM antibody is a humanmonoclonal anti-PIM6 antibody that binds specifically to at least onepolymannose structure in PIM6 and/or in the PIM6 crossreactive mannandomain of LAM. In some embodiments, the first anti-LAM antibody and thesecond anti-LAM antibody, or the anti-PIM6/LAM antibody are of differentisotypes. In some embodiments, at least one of the first anti-LAMantibody and the second anti-LAM antibody, and the anti-PIM6/LAMantibody are recombinant antibodies. In other embodiments, neither thefirst anti-LAM antibody nor the second anti-LAM antibody, nor theanti-PIM6/LAM antibody, are recombinant antibodies. In yet otherembodiments, both the first anti-LAM antibody and the second anti-LAMantibody, or the anti-PIM6/LAM antibody, are recombinant antibodies. Infurther embodiments, the method includes administration of at least oneantibiotic. In such embodiments, the at least one antibiotic can beadministered (e.g., co-administered) simultaneously with the first andsecond antibodies, or the at least one antibiotic can be administered ata time point different from the time point of administration of thefirst and second antibodies. In some embodiments, the infection isactive. In other embodiments, the infection is latent. In someembodiments, the infection is a multiple-drug resistant (MDR)tuberculosis infection. In other embodiments, the infection is anextensively-drug resistant (XDR) tuberculosis infection.

In some embodiments, the present invention provides vaccines orpharmaceutical compositions for preventing infection by a virulentmember of the Mycobacterium tuberculosis-complex. In some embodiments,the invention provides a method of stimulating a host immune response ina patient including administering to said patient a therapeuticallyeffective amount of a LAM antigen and/or a PIM6 antigen. In someembodiments these antigens are conjugated to immunogenic proteincarriers, and/or are co-administered with an adjuvant that potentlystimulates an immune response to glycolipid antigens. In someembodiments, the vaccine or pharmaceutical composition induces ananti-LAM antibody that specifically binds to a Man-LAM epitope, and/oran anti-PIM6/LAM antibody that specifically binds to at least onepolymannose structure in the PIM6 mannan domain. In further embodiments,the Man-LAM epitope present in the vaccine or pharmaceuticalcompositions includes di-mannose or tri-mannose capped Ara4 and/or Ara6structures. In yet further embodiments, the Man-LAM epitope isdi-mannose capped Ara6. In some embodiments, the Man-LAM epitope has atleast one MTX substitution. In some embodiments, the anti-LAM antibodyand/or anti-PIM6/LAM antibody is an IgM antibody. In other embodiments,the anti-LAM antibody and/or anti-PIM6/LAM antibody is a recombinantantibody.

In some embodiments, the present invention provides a method ofpreventing infection by a virulent member of the Mycobacteriumtuberculosis-complex in an individual by passive administration of aprotective antibody. In some embodiments, the anti-LAM antibody is ahuman monoclonal anti-LAM antibody that binds specifically to one ofAra4 and Ara6 or combinations thereof. In some embodiments, theanti-PIM6/LAM antibody is a human monoclonal anti-PIM6/LAM antibody thatbinds specifically to at least one polymannose structure in the PIM6 andLAM mannan domain. In some embodiments, the method includesadministering to an individual a therapeutically effective amount of ananti-LAM antibody that specifically binds to a Man-LAM epitope, and/oran anti-PIM6 antibody that specifically binds to a PIM6 epitope (e.g.,an epitope shared by PIM6 and LAM). In further embodiments, the targetedManLAM epitope includes di-mannose or tri-mannose capped Ara4 and/orAra6 structures. In yet further embodiments, the ManLAM epitope isdi-mannose capped Ara6. In some embodiments, the ManLAM eptiope has atleast one MTX substitution. In some embodiments, the anti-LAM antibodyor anti-PIM6/LAM antibody is an IgM antibody. In other embodiments, theanti-LAM antibody or anti-PIM6/LAM antibody is a recombinant antibody.

In some embodiments, the present invention provides passiveadministration of a protective antibody via recombinant vectors. In someembodiment, the recombinant vectors include a first nucleic acidencoding for an IgVL of an anti-LAM antibody and a second nucleic acidencoding an IgVH of an anti-LAM antibody, wherein each of the nucleicacids is operably linked to a promoter region. In some embodiments, atleast one of the IgVL and IgVH is derived from a human monoclonalanti-LAM antibody that binds specifically to one of Ara4 and Ara6 orcombinations thereof. In other embodiment, the recombinant vectorsinclude a first nucleic acid encoding for an IgVL of an anti-PIM6/LAMantibody and a second nucleic acid encoding an IgVH of an anti-PIM6/LAMantibody, wherein each of the nucleic acids is operably linked to apromoter region. In some embodiments, the recombinant vectors includeadditional transcriptional regulation elements. In some embodiments, atleast one of the first nucleic acid sequence and the second nucleic acidsequence are organized in an operon. In some embodiments, at least oneof the first nucleic acid sequence and the second nucleic acid sequenceare organized in an expression cassette. In some embodiments, the firstnucleic acid sequence and the second nucleic acid sequence are organizedin a single expression cassette. In some embodiments, the first nucleicacid and the second nucleic acid are located in the same cloning vector.In other embodiments, the first nucleic acid and the second nucleic acidare located in different cloning vectors. In some embodiments,expression of the first nucleic acid and the second nucleic acid may beconcomitant. In other embodiments, expression of the first nucleic acidand the second nucleic acid is separably inducible. In some embodiments,expression of the first nucleic acid may be temporally separate fromexpression of the second nucleic acid. In some embodiments, therecombinant vector is a plasmid. In other embodiments, the recombinantvector is a non-replicated virus. In further embodiments, therecombinant vector is an adeno-associated virus.

In some embodiments, the present invention provides for a method oftreating infection by a virulent member of the Mycobacteriumtuberculosis-complex in an individual. In some embodiments, the methodincludes administering to an individual a first nucleic acid coding foran IgVH of an anti-LAM antibody, and a second nucleic acid coding for anIgVL of an anti-LAM antibody, wherein each of the nucleic acids isoperably linked to a promoter region. In other embodiments, the methodincludes administering to an individual a first nucleic acid coding foran IgVH of an anti-PIM6/LAM antibody, and a second nucleic acid codingfor an IgVL of an anti-PIM6/LAM antibody, wherein each of the nucleicacids is operably linked to a promoter region. In some embodiments, atleast one of the IgVL and IgVH is derived from a human monoclonalanti-LAM antibody that binds specifically to one of Ara4 and Ara6 orcombinations thereof, or from a human monoclonal anti-PIM6/LAM antibodythat binds specifically to at least one polymannose structure in thePIM6 mannan domain. In some embodiments, the first nucleic acid and thesecond nucleic acid are located in the same cloning vector. In otherembodiments, the first nucleic acid and the second nucleic acid arelocated in different cloning vectors. In some embodiments, therecombinant vector is a plasmid. In other embodiments, the recombinantvector is a non-replicated virus. In further embodiments, therecombinant vector is an adeno-associated virus.

Additional embodiments, features and advantages will be readily apparentto one of skill in the art based on the disclosure provided herein.Other features will become more apparent to persons having ordinaryskill in the art to which the package pertains and from the followingdescription and claims. Although antibodies, compositions, kits andmethods similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable antibodies,compositions, kits and methods are described below. All publications,patent applications, and patents mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. The particularembodiments discussed below are illustrative only and not intended to belimiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A—Model of IgG form of A194-01 and fragments thereof used inbinding competition assays. These included monovalent scFv and Fabstructures, and divalent scFv dimer and natural IgG. FIG. 1B—Competitioncurves showing that the monovalent forms of A194-01 competed lesseffectively than the divalent forms. 1C—Structure of higher-valent formof A194-01. This represents a homologous tetravalent A194-01 scFv-IgG,which contains an A194-01 scFv domain joined to the N-terminus of eachof the normal heavy chains.

FIG. 2A—Binding activity of P30B9 IgG and IgM forms, and IgM in whichthe 6 amino acid insert in the VH region was deleted, or the 9 somaticmutatic mutations in the VH region were reverted to the nearestgerm-line sequence, to ManLAM derived from Mycobacterium tuberculosis.The 6 amino acid insert contributed to a greater extent than the 9somatic acid mutations to reactivity. FIGS. 2B and 2C compare thereactivity of the P30B9 IgM and IgG forms and the mutation with the 6amino acid deletion in the heavy chain against ManLAM from Mycobacteriumtuberculosis (FIG. 2B) and PILAM from Mycobacterium smegmatis (FIG. 2C).The IgM form, but not the IgG form, reacted specifically with ManLAMderived from Mycobacterium tuberculosis (FIG. 2B) but not PILAM (FIG.2C), and the reactivity of the A6 amino acid mutant was highly reducedfor ManLAM and negative for PILAM.

FIG. 3 —Comparing the reactivity of 2 human mAbs and 4 mouse mAbs vsPILAM in the left panel and vs. ManLAM isolated from the H37Rv strain ofMycobacterium tuberculosis in the right panel. Curves were plotted usingthe molar concentrations of the antibodies, to control for the differentmolecular weights of these reagents

FIG. 4A—Structures of 25 synthetic oligosaccharides representingmicrobial glycan structures related to motifs present in LAM. Thesestructures were coupled to BSA carrier protein and used to probe epitopespecificity. FIG. 4B—Binding profiles for six LAM-specific monoclonalantibodies against panel of 25 synthetic oligosaccharides. Bindingresults are shown for three concentrations, and the relative affinitiesof the antibodies to these antigens is indicated by the titrationpattern.

FIG. 5 —Left hand panel—structure of IgA1 (FIG. 5A), IgA2 (FIG. 5B) anddimeric IgA1-J dimeric complex (FIG. 5C). Right hand panel—SDS-PAGE gelof purified P30B9 IgA1, IgA2 and IgA3 proteins, both before and afterreduction with DTT. P30B9 IgA3 was later revealed to be an artifact ofPCR with a longer hinge region.

FIG. 6 —Binding curves of different isotypes of P30B9 to ManLAM showinggreatest activity for IgM form followed by IgA forms, with no reactivityfor the IgG form.

FIG. 7 —Comparison of efficiency of biotinylated monoclonal antibodyprobes at detecting soluble ManLAM in CS-35 capture assay, in which theindicated concentration of ManLAM was captured by CS-35 and detected bythe indicated mAbs labeled with biotin.

FIG. 8 —Binding curves of P30B9 to various mannose-capped Ara4structures, or to tetra- and penta-mannose structures. Preferentialbinding was seen for structures 3 (dimannose-Ara4) and 59, whichcontained the related α-Manp(1□2)-Manp linkage.

FIG. 9 —Titration of monoclonal anti-LAM antibodies against variousuncapped LAM-related glycoconjugates to determine structuralrequirements for reactivity. FIG. 9A—Analysis of the importance of theAra-α(1□5)-Ara linkage at the penultimate position from the non-reducingend of the Ara4 sequence. FIG. 9B—Analysis of the dependence of theAra-β(1□2)-Ara linkage at the terminal position of the Ara4 sequence.

FIG. 10 Binding curves of A194-01 IgG and three murine anti-LAMantibodies against various Ara6-containing glycoconjugates, showing theeffect of different capping motifs on antibody reactivity.

FIG. 11 Binding competition studies to measure the ability of individualanti-LAM antibodies to compete for binding of a probe antibody to theManLAM antigen. Antibodies were biotinylated when tested againstantibodies from the same species. Note inability of A194-01 to competefor biotinylated P30B9.

FIG. 12 —Competition of binding of anti-LAM monoclonal antibodies to LAMderived from Mycobacterium tuberculosis (ManLAM) and LAM derived fromMycobacterium smegmatis (PTLAM). Efficient competition between FIND25and P30B9 for ManLAM is consistent with predominance ofdimannose-substituted Ara6, while lack of competition of these two mAbsby A194 is consistent with its poor reactivity with dimannose cappedstructures. The efficient competition of A194 for FIND25 vs PILAM isconsistent with the absence of dimannose capping in this structure.

FIG. 13 —Binding competition of biotinylated probe monoclonal antibodieswith excess unmodified antibodies against natural LAM antigens andselected glycoconjugates. FIG. 13A-Competition of binding ofbiotinylated A194-01 IgG, CS-35 and FIND25 to MAnLAM by four mAbs; FIG.13B—Competition of binding of FIND25 to both ManLAM and PILAM by threemAbs; FIG. 13C—Competition of binding of P30B9 IgM to MAnLAM and twosynthetic glycoconjugate antigens by four mAbs.

FIG. 14 —Engineered variants and/or derivatives of A194-01 react with abroader range of glycoconjugates, including di- and tri-mannosesubstituted forms poorly recognized by the IgG isotype of A194-01.

FIG. 15 —Differential competition of A194-01 IgG and engineered variantsand/or derivatives of A194-01 for binding of FIND25 and P30B9 IgM toManLAM. Although A194 IgG doesn't compete with P30B9 or FIND25 againstManLAM, the multimeric forms do compete, consistent with the broaderepitope specificity of these forms. As shown above, A194 IgG doescompete well with FIND25 for PILAM.

FIG. 16 —Comparison of analysis of the effect of mannose-capping on thereactivity of CS-40, A194-01 and P30B9 mAbs. Antibody bindingspecificities were measured by ELISA against specific glyconjugatescontaining different mannose substitutions. The antibody titrations andthe structures of the mannose-containing glycan antigens are shown.

FIG. 17A, 17B, 17C. Homologous scFv-IgG. In this example, both the IgGand scFv domains are derived from the same antibody. This results in anincreased valency (tertavalent vs. divalent) but does not directlymodify the target specificity. FIG. 17B. Heterologous scFv-IgG. Inaddition to the increase in valency, there is also a broadenedspecificity introduced, which may allow recognition of distinct epitopesin a single antigen molecule. FIG. 17C. Heterologous scFv-IgM. In thisformulation a distinct scFv is combined with an IgM construct. Oneexample would be joining of the A194-01 scFv with the P30B9 IgM. Inaddition to the increase in valency, this would introduce an additionalepitope specificity, which may allow multivalent recognition of distinctepitopes that may not be recognized by the homologous scFv-IgM, and leadto increased affinities.

FIG. 18A-18C—Mapping of epitopes recognized by new mAbs. The epitopespecificity of P95C1 was compared to that of two previously describedmAbs, A194-01 and P30B9, and two new mAbs, P61H5 and P83A8, thatrecognize epitopes related to those two previously described mAbs. FIG.18A. Reactivity of LAM-specific mAbs for LAM precursor molecules. P30B9and P61H5 were specific for ManLAM over PILAM, while A194-01, P83A8 andP95C1 recognized both forms of LAM. P95C1 also bound efficiently with LMand PIM6. The weak reactivity of the other mAbs for LM and PIM6 is dieat least in part, to contamination of these materials by ManLAM. FIG.18B. Reactivity of synthetic LAM-derived glycoconjugates. FIG. 18C. Incontrast to previously known mAbs, P95C1 was the only antibody that didnot recognize any of the polyarabinose structures, but reactedspecifically with two poly-mannose structures, YB-BSA-05 and YB-BSA-11,that resembled structures present in PIM6 and in the mannan domains atthe base of LM and LAM.

FIG. 19 —Effect of isotype switching on binding of P95C1 and P30B9 toManLAM and PI-LAM. For P95C1, IgM, IgA and IgG isotypes all havecomparable binding activity for both ManLAM and PILAM, unlike P30B9which react only with ManLAM and only in IgM and IgA forms but not asIgG.

FIG. 20A-20B—Western blot analysis of crossreactivity of P95C1 with LAMand additional M.tb glycolipids. FIG. 20A Purified LAM associatedglycolipids were separated on 12% SDS-PAGE gel followed by oxidation andstaining of sugar molecules with periodic acid-Schiff stain, to revealmaterial containing reactive glycans. FIG. 20B Parallel blots wereprobed with mAbs A194 IgG1, P30B9 IgM, and P95C1 IgM followed byalkaline phosphatase conjugated anti human IgG and IgM secondaryantibodies and treatment with bcip/nbt color development substrate.A194-01 crossreacts with ManLAM from M.tb and PILAM from M.smeg. P30B9is specific for M.tb ManLAM. P95C1 recognizes both species of LAM, aswell as LM and PIM6 isolated from M.tb. Weak staining by A194-01 ofbands in LM and PIM6 that co-migrate with LAM is apparently due to minorcontamination of these samples with LAM.

FIG. 21 —Alignments of amino acid sequences for A194 heavy chain andlight chain variable regions sequences and their comparison with theirclosest germline sequences. In the top alignment, from the top, thefirst amino acid sequence (A194-VH) is an A194 heavy chain variableregion sequence without the CDR3 sequence (SEQ ID NO:23). The heavychain variable region sequence without CDR3 is SEQ ID NO:21. In the topalignment, the second amino acid sequence (germline Homsap IGHV3-20*01)is SEQ ID NO:22. In the top alignment, the third amino acid sequence isthe CDR3 of a A194 heavy chain variable region and is SEQ ID NO:23. Inthe bottom alignment, from the top, the first amino acid sequence(A-194-Vk) is an A194 light chain variable region without the CDR3sequence (SEQ ID NO: 26). The light chain variable region sequencewithout CDR3 is SEQ ID NO:24. In the bottom alignment, the second aminoacid sequence (germline Homsap IGKV3-15*01) is SEQ ID NO:25. In thebottom alignment, the third sequence is the CDR3 of a A194 light chainvariable region and is SEQ ID NO:26.

FIG. 22 —Amino acid sequences for P30B9-IgM heavy chain and light chainvariable region sequences and their comparisons with their closestgermlines. In the top alignment, from the top, the first amino acidsequence (P30B9-Vh) is a P30B9-IgM heavy chain variable region sequencewithout the CDR3 sequence (SEQ ID NO:29). The heavy chain variableregion sequence without CDR3 is SEQ ID NO: 27. The second amino acidsequence (Homsap IGHV4-34*01 F) is SEQ ID NO:28. The third amino acidsequence is of a P30B9-IgM heavy chain variable region and is SEQ IDNO:29. In the bottom alignment, from the top, the first amino acidsequence (P30B9-Vk) is a P30B9 light chain variable region without theCDR3 sequence (SEQ ID NO:32). The light chain variable region sequencewithout CDR3 is SEQ ID NO:30. In the bottom alignment, the second aminoacid sequence (germline Homsap IGKV1-5*03) is SEQ ID NO:31. In thebottom alignment, the third sequence is the CDR3 of a P30B9 light chainvariable region and is SEQ ID NO:32.

FIG. 23 —Alignments of amino acid sequences for P95C1-IgM heavy chainand light chain variable regions sequences and their comparison withtheir closest germline sequences. In the top alignment, from the top,the first amino acid sequence (P95C1-VH) is an P95C1 heavy chainvariable region sequence without the CDR3 sequence (SEQ ID NO:18). Theheavy chain variable region sequence without CDR3 is SEQ ID NO:33. Inthe top alignment, the second amino acid sequence (germline HomsapIGHV4-4*02) is SEQ ID NO:34. In the top alignment, the third amino acidsequence is the CDR3 of a P95C1-gM heavy chain variable region and isSEQ ID NO:18. In the bottom alignment, from the top, the first aminoacid sequence (P95C1-Vk) is a P95C1 light chain variable region withoutthe CDR3 sequence (SEQ ID NO:15). The light chain variable regionsequence without CDR3 is SEQ ID NO:36. In the bottom alignment, thesecond amino acid sequence (germline Homsap IGKV4-1*01 F) is SEQ IDNO:37. In the bottom alignment, the third sequence is the CDR3 of aP95C1 light chain variable region and is SEQ ID NO:15.

DETAILED DESCRIPTION A. Definitions

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

An anti-LAM antibody may take one of numerous forms in the art, asdisclosed herein. Antibodies are in part defined by the antigens towhich they bind, thus, an “anti-LAM antibody” is any such antibody whichspecifically binds at least one epitope of lipoarabinomannan (LAM) asdescribed herein. It is understood in the art that an antibody is aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen binding portionthereof. A heavy chain is comprised of a heavy chain variable region(VH) and a heavy chain constant region (CH1, CH2 and CH3). A light chainis comprised of a light chain variable region (VL) and a light chainconstant region (CL). The variable regions of both the heavy and lightchains comprise framework regions (FWR) and complementarity determiningregions (CDR). The four FWR regions are relatively conserved while CDRregions (CDR1, CDR2 and CDR3) represent hypervariable regions and arearranged from NH2 terminus to the COOH terminus as follows: FWR1, CDR1,FWR2, CDR2, FWR3, CDR3, FWR4. The variable regions of the heavy andlight chains contain a binding domain that interacts with an antigenwhile, depending of the isotype, the constant region(s) may mediate thebinding of the immunoglobulin to host tissues or factors.

An anti-PIM6/LAM antibody may take one of numerous forms in the art, asdisclosed herein. An “anti-PIM6/LAM antibody” is any such antibody whichspecifically binds at least one epitope that is shared byphosphatidylinositol mannoside 6 (PIM6) and LAM as described herein. Ahuman mAb specific for an epitope shared by LAM and PIM6 describedherein is P95C1 which binds specifically to at least one polymannosestructure in PIM6 and in the PIM6 related mannan domain of LM and LAM.P95C1 binds to both LAM and PIM6 because it sees a common (shared)epitope, and is thus referred to herein as an “anti-PIM6/LAM antibody”or “anti-PIM6/LAM monoclonal antibody,” “human anti-PIM6/LAM antibody”or “human anti-PIM6/LAM monoclonal antibody.”

It is known in the art that it is possible to manipulate monoclonal andother antibodies and use techniques of recombinant DNA technology toproduce other antibodies or chimeric molecules which retain thespecificity of the original antibody. Such techniques may evolveintroducing DNA encoding the immunoglobulin variable region, or CDRs, ofan antibody to the constant regions, or constant regions plus frameworkregions, of a different immunoglobulin.

The term “antibody” (Ab) as used herein is used in the broadest senseand specifically may include any immunoglobulin, whether natural orpartly or wholly synthetically produced, including but not limited tomonoclonal antibodies, polyclonal antibodies, multispecific antibodies(for example, bispecific antibodies and polyreactive antibodies), andantibody fragments. Thus, the term “antibody” as used in any contextwithin this specification is meant to include, but not be limited to,any specific binding member, immunoglobulin class and/or isotype (e.g.,IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE) andbiologically relevant fragment or specific binding member thereof,including but not limited to Fab, F(ab′)₂, scFv (single chain or relatedentity) and (scFv)₂.

The term “antibody fragments” as used herein may include those antibodyfragments obtained using techniques readily known and available to thoseof ordinary skill in the art, as reviewed herein. Therefore, the term“antibody” describes any polypeptide or protein comprising a portion ofan intact antibody, such as the antigen binding or variable region ofthe intact antibody. These can be derived from natural sources, or theymay be partly or wholly synthetically produced. Examples of antibodyfragments include, but are not limited to, Fab, Fab′, F(ab′)2, and Fvfragments; diabodies, and linear antibodies. In particular, as usedherein, “single-chain Fv” (“sFv” or “scFv”) are antibody fragments thatcomprise the VH and VL antibody domains connected into a singlepolypeptide chain. The sFv polypeptide can further comprise, e.g., alinker such as a flexible polypeptide linker between the VH and VLdomains that enables the scFv to form the desired structure for antigenbinding.

The term “monoclonal antibody” or “mAb” as used herein may refer to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts.

The terms “variants,” “derivatives,” and/or “variants and/orderivatives” as used herein may refer to antibodies, antibody fragments,recombinant antibodies, whether derived from natural sources or partlyor wholly synthetically produced, as well as proteins, proteinfragments, and polypeptides, inasmuch as the foregoing compounds areeither structurally similar, i.e. retain a degree of identity that is atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 80%, at least 85%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, or greater sequence identity with anoriginal unmodified antibody, and/or, independent of structuralidentity, may be functionally similar to the original unmodifiedanti-LAM and anti-PIM6/LAM antibodies, that is, they retain the abilityto specifically bind to at least one epitope of LAM or to the sharedPIM6/LAM epitope, respectively. For example, such variants and/orderivatives may include anti-LAM or anti-PIM6/LAM antibodies withvariant Fc domains, chimeric antibodies, fusion proteins, bispecificantibodies, or other recombinant antibodies. Such variants and/orderivative antibodies may, but not necessarily, possess greater bindingspecificity for one or more epitope(s) of LAM, or PIM6, and/or may beable to bind to additional LAM or PIM6 epitopes.

The term “biological sample” refers to a sample obtained from anorganism (e.g., patient) or from components (e.g., cells) of anorganism. The sample may be of any biological tissue, cell(s) or fluid.The sample may be a “clinical sample” which is a sample derived from asubject, such as a human patient. Such samples include, but are notlimited to, saliva, sputum, blood, blood cells (e.g., white cells),amniotic fluid, plasma, semen, bone marrow, and tissue or fine needlebiopsy samples, urine, peritoneal fluid, and pleural fluid, or cellstherefrom. Biological samples may also include sections of tissues suchas frozen sections taken for histological purposes. A biological samplemay also be referred to as a “patient sample.” A biological sample mayalso include a substantially purified or isolated protein, membranepreparation, or cell culture.

The terms “effective amount” or “therapeutically effective amount” asused herein may refer to an amount of the compound or agent that iscapable of producing a medically desirable result in a treated subject.The treatment method can be performed in vivo or ex vivo, alone or inconjunction with other drugs or therapy. A therapeutically effectiveamount can be administered in one or more administrations, applicationsor dosages and is not intended to be limited to a particular formulationor administration route.

The term “antigen binding fragment” or “Fab” as used herein may refer toa region on an antibody that binds to antigens. One of ordinary skill inthe art will understand that Fabs are comprised of one constant and onevariable domain of each of the heavy and light chain of an antibody.

As used herein, the terms “specific binding,” “selective binding,”“selectively binds,” and “specifically binds,” may refer to antibodybinding to an epitope on a predetermined antigen but not to otherantigens. Typically, the antibody (i) binds with an equilibriumdissociation constant (K_(D)) of approximately less than 10⁻⁶ M, such asapproximately less than 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lowerwhen determined by, e.g., surface plasmon resonance (SPR) technology ina BIACORE® 2000 surface plasmon resonance instrument using thepredetermined antigen, e.g., a LAM epitope, as the analyte and theantibody as the ligand, or Scatchard analysis of binding of the antibodyto antigen positive cells, and (ii) binds to the predetermined antigenwith an affinity that is at least two-fold greater than its affinity forbinding to a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen.

The terms “conservative sequence modifications” or “conservativesubstitutions” as used herein may refer to amino acid modifications thatdo not significantly affect or alter the binding characteristics of theantibody containing the amino acid sequence. Such conservativemodifications include amino acid substitutions, additions and deletions.Modifications can be introduced into an antibody of the invention bystandard techniques known in the art, such as site-directed mutagenesisand PCR-mediated mutagenesis. Conservative amino acid substitutions areones in which the amino acid residue is replaced with an amino acidresidue having a similar side chain. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine), beta-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). Thus, one or more amino acid residues within theCDR regions of an antibody of the invention can be replaced with otheramino acid residues from the same side chain family and the alteredantibody can be tested for retained function using the functional assaysdescribed herein.

The term “identity” as used herein may refer to the existence of sharedstructure between two compositions. The term “identity” in the contextof proteins may refer to the amount (e.g. expressed in a percentage) ofoverlap between two or more amino acid and/or peptide sequences. In thecontext of nucleic acids, the term may refer to the amount (e.g.expressed in a percentage) of overlap between two or more nucleic acidsequences. As used herein, the percent (%) identity between twosequences is equivalent to the percent identity between the twosequences. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e., %identity=# of identical positions/total # of positions×100), taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences. The comparison ofsequences and determination of percent identity between two sequencescan be accomplished using a mathematical algorithm. Such identity iswell-represented in the art via local alignment tools and/or algorithms,and may include pairwise alignment, multiple sequence alignment methods,structural alignment methods, and/or phylogenetic analysis methods.Specific examples include the following. The percent identity betweentwo amino acid sequences can be determined using the algorithm of E.Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which hasbeen incorporated into the ALIGN program (version 2.0), using a PAM120weight residue table, a gap length penalty of 12 and a gap penalty of 4.In addition, the percent identity between two amino acid sequences canbe determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453(1970)) algorithm which has been incorporated into the GAP program inthe GCG software package (available at www.gcg.com), using either aBlossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12,10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Additionallyor alternatively, the protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the XBLAST program (version 2.0) of Altschul, et al.(1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the antibody molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used.

The terms “co-administration,” “co-administered,” and “in combinationwith” as used herein may refer to the administration of at least twoagents or therapies to a subject. In some embodiments, theco-administration of two or more agents/therapies is concurrent. Inother embodiments, a first agent/therapy is administered prior to asecond agent/therapy. Those of skill in the art understand that theformulations and/or routes of administration of the variousagents/therapies used may vary.

The term “carriers” as used herein may include pharmaceuticallyacceptable carriers, excipients, or stabilizers that are nontoxic to thecell or mammal being exposed thereto at the dosages and concentrationsemployed. Often the physiologically acceptable carrier is an aqueous pHbuffered solution. Examples of physiologically acceptable carriersinclude, but not limited to, buffers such as phosphate, citrate, andother organic acids; antioxidants including, but not limited to,ascorbic acid; low molecular weight (less than about 10 residues)polypeptide; proteins, such as, but not limited to, serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such as, but notlimited to, polyvinylpyrrolidone; amino acids such as, but not limitedto, glycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including, but not limited to,glucose, mannose, or dextrins; chelating agents such as, but not limitedto, EDTA; sugar alcohols such as, but not limited to, mannitol orsorbitol; salt-forming counterions such as, but not limited to, sodium;and/or nonionic surfactants such as, but not limited to, TWEEN.;polyethylene glycol (PEG), and PLURONICS.

The term “treating” or “treatment” of a disease refers to executing aprotocol, which may include administering one or more drugs to a patient(human or otherwise), in an effort to alleviate signs or symptoms of thedisease. Alleviation can occur prior to signs or symptoms of the diseaseappearing as well as after their appearance. Thus, “treating” or“treatment” includes “preventing” or “prevention” of disease. The terms“prevent” or “preventing” refer to prophylactic and/or preventativemeasures, wherein the object is to prevent or slow down the targetedpathologic condition or disorder. For example, in the case of infectionby a virulent strain of the Mycobacterium tuberculosis-complex,“preventing” or “preventing” may arise in a situation where a course oftreatment is advanced in order to prevent or stall infection by avirulent strain of the Mycobacterium tuberculosis-complex, such asthrough vaccination or passive administration of a protective antibody.Such “preventing” or “prevention” also arise in the case of latentinfection by Mycobacterium tuberculosis, in which the object would be toprevent active infection and/or clear a patient of said latentinfection. In addition, “treating” or “treatment” does not requirecomplete alleviation of signs or symptoms, does not require a cure, andspecifically includes protocols that have only a marginal effect on thepatient.

The terms “patient,” “subject” and “individual” are used interchangeablyherein and may refer to a biological system to which a treatment can beadministered. A biological system can include, for example, anindividual cell, a set of cells (e.g., a cell culture), an organ, atissue, or a multi-cellular organism. A “patient,” “subject” or“individual” can refer to a human patient, subject or individual or anon-human patient, subject or individual.

The term “epitope” as used herein may refer to the region of an antigento which an antibody or T cell binds. An “antigen” refers to a substancethat elicits an immunological reaction or binds to the products of thatreaction.

As used herein, the term “vector” means a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked.Vectors capable of directing the expression of genes to which they areoperatively linked are referred to herein as “expression vectors.”

As used herein, “protein” and “polypeptide” are used synonymously tomean any peptide-linked chain of amino acids, regardless of length orpost-translational modification, e.g., glycosylation or phosphorylation.

The term “labeled,” with regard to an antibody, nucleic acid, peptide,polypeptide, cell, or probe, is intended to encompass direct labeling ofthe antibody, nucleic acid, peptide, polypeptide, cell, or probe bycoupling (i.e., physically linking) a detectable substance to theantibody, nucleic acid, peptide, polypeptide, cell, or probe.

The terms “purified” or “isolated” peptide, polypeptide, or proteinrefers to a peptide, polypeptide, or protein, as used herein, may referto a peptide, polypeptide, or protein that has been separated from otherproteins, lipids, and nucleic acids with which it is naturallyassociated. The polypeptide/protein can constitute at least 10% (i.e.,any percentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%, 60%, 70%,80%, 85%, 90%, 95%, and 99%) by dry weight of the purified preparation.Purity can be measured by any appropriate standard method, for example,by column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis. An isolated polypeptide/protein (e.g., anti-LAM antibodies)described in the invention can be produced by recombinant DNAtechniques.

B. Mycobacterium tuberculosis

Tuberculosis (TB) remains one of the world's deadliest communicablediseases, currently infecting approximately one-third of the world'spopulation. An estimated 9.0 million people developed TB in 2013, and anestimated 1.5 million people died from the disease. Although therecurrently are antibiotic treatments available, these require lengthytreatments, and are increasingly compromised by the development ofmulti-drug resistant (MDR-TB) strains, which currently are responsiblefor about 3.5% of recent infections. These strains are much harder totreat and have significantly poorer cure rates. Also spreading areextensively drug-resistant TB (XDR-TB) strains, which are even moreexpensive and difficult to treat than MDR-TB strains, and have now beenreported in 100 countries around the world.

There is a long-established paradigm that immunity against TB reliessolely on cellular defense mechanisms. However, studies in the HIV fieldhighlight the remarkable ability of the human humoral immune system togenerate diverse antibodies with remarkable neutralization breadth andpotency, and the present invention highlights the ability of the humoralimmune system to produce high affinity antibodies that recognizemultiple LAM epitopes. This suggests that much of the past difficulty indemonstrating an important role for antibody-mediated protection againstTB may be due to the limitations in the quality and source of theantibodies used in past studies, and that applying methods of thepresent invention to generate more highly evolved antibodies fromchronically-infected human patients may illustrate the critical role ofthe humoral response in immunity to TB.

Some embodiments of the invention are directed to methods for the invitro culture of memory B cells from infected humans and molecularcloning of the variable regions of IgG heavy (H) and light (L) chainsfrom a single cell. These methods may be utilized to generate humanmonoclonal antibodies against the major surface antigen LAM. The presentinvention relates to such antibodies, and engineered derivatives ofthese antibodies, that possess unique epitope specificities and bindingproperties, and the immunodiagnostic and immunotherapeutic applicationsof these antibodies.

C. Lipoarabinomannan (LAM)

One prominent antigenic target of the antibodies of the presentinvention is the surface glycolipid, lipoarabinomannan (LAM), a majorstructural component of the cell wall of Mycobacteriumtuberculosis-complex members. The present invention identifies apreviously unappreciated heterogeneity in the antigenic structure of LAMand in the humoral immune response towards LAM in response to infectionand immunization. The structure of LAM is detailed in Khoo et al.,“Variation in Mannose-capped Terminal Arabinan Motifs ofLipoarabinomannan from Clinical Isolates of Mycobacterium tuberculosisand Mycobacterium avium Complex,” Journal of Biological Chemistry Vol276, No. 6, Feb. 9, 2001, incorporated by reference herein in itsentirety. The structure of LAM is complex, exhibiting an overalltripartite structure with four distinct structural domains; aphosphatidylinositol lipid anchor (Mannonsyl-Phosphatidyl-myo-Inositol),an α(1→6)-linked D-mannan backbone with terminal α(1→2)-Manp-linked sidechains, an D-arabinan chain containing multipletetra-/hexa-arabinofuranoside branches, and various capping motifs. LAMconsists of a heterogeneous population of molecules, which can beresolved into multiple isoforms that possess different biologicalproperties. This heterogeneity is due to varying lengths of the mannanand arabino chains, different branching patterns, different numbers ofsuch branches, and modification of the arabino-side chains by mannosecapping, MTX addition and acylation by fatty acids, succinates andlactates.

Virulent strains of the Mycobacterium tuberculosis-complex areextensively capped with mono-, di-, and tri-α(1→2)-D-Manp saccharideunits, while fast growing non-pathogenic strains like M. smegmatis haveuncapped ends or phosphatidyl-myo-inositol caps (PILAM). It has beenestimated that 40-70% of the nonreducing termini of LAM from pathogenicstrains of the Mycobacterium tuberculosis-complex are mannose-capped,and analysis of the relative abundance of the different cap motifs forthe virulent MT103 clinical strain showed that the dimannosyl unit wasthe major structural motif (75-80%), while the mannosyl and thetrimannosyl motifs were present at lower concentrations (10-13%). Thisextensive capping may present a unique marker to differentiate virulentstrains of the Mycobacterium tuberculosis-complex fromnon-virulent/non-pathogenic strains, such as M. smegmatis, and may alsoprovide potential antigenic targets for therapeutic use of the anti-LAMantibodies of the present invention. In addition, some of the terminalmannose sugars in ManLAM found in the strain M. tuberculosis are furthermodified by α(1□4) addition of a unique structure,5-deoxy-5-methyl-thio-pentofuranose (MTX), which affects theimmunoreactivity towards different mAbs sensitive to capping motifs,such as A194-01 and P30B9; MTX addition increases reactivity withA194-01 and decreases reactivity towards P30B9. This substitution ispresent in low abundance, and may present a unique marker to identify M.tuberculosis, potentially even from other virulent members of theMycobacterium tuberculosis-complex, such as from M. bovis and M.africanum, and may also provide a potential antigenic target fortherapeutic use of the anti-LAM antibodies of the present invention.

Secreted forms of LAM are important targets for immunodiagnostic assaysof infection by pathogenic members of the M. tuberculosis-complex. Inaddition, a considerable body of evidence indicates that LAM is animportant mediator of a number of functions that promote productiveinfection and pathogenicity. LAM is involved in maintaining cell wallintegrity and resistance to beta-lactam antibiotics. Reduced expressionof LAM on the bacterial surface correlated with defective macrophageentry, inhibition of phagosome-lysosome fusion, attenuation inmacrophages, and increased sensitivity to adaptive immunity, and thebinding of terminal mannosyl units of ManLAM to the mannose receptor onthe surface of macrophages has been described as a critical step in theuptake of mycobacteria into phagocytic cells. Without wishing to bebound by theory, it is believed that ManLAM interacts with the C-typelectins, such as dendritic cell-specific intercellular adhesionmolecule-3 (ICAM-3) grabbing non-integrin (DC-SIGN) the macrophagemannose receptor (MMR) and Dectin-2 on dendritic cells. Once insidemacrophages, LAM is believed to inhibit phagosome-lysosome fusion whichwould lead to the destruction of the bacteria, thereby allowing thebacteria to persist inside the macrophages.

LAM is also secreted from the surface of bacteria, and the extracellularLAM binds to dendritic cell-surface receptors, including DC-SIGN andDectin-2. These interactions are believed to suppress dendritic cellfunction and interfere with the host immune system, contributing toimmune evasion. Because LAM is in relatively large quantities duringactive infection, it can be detected in the blood and urine of infectedpatients, for example, by one or more anti-LAM antibodies of the presentinvention. These may be used, for example, in diagnostic kits andmethods related to said diagnostic kits.

D. Anti-LAM and Anti-PIM6/LAM Antibodies

The anti-LAM antibodies of the present invention may comprise isolated,cultured, or engineered variants and/or derivatives of human monoclonalantibodies that recognize at least one epitope on lipoarabinomannan(LAM). An anti-PIM6/LAM antibody (e.g., P95C1) as described hereinspecifically binds at least one polymannose structure in PIM6 and in thePIM6 cross-reactive mannan domain of LAM. The anti-LAM and anti-PIM6/LAMantibodies of the present invention may be purified according to methodsknown in the art. Such methods may include, for example but not limitedto, affinity chromatography, ion exchange chromatography, immobilizedmetal chelate chromatography, thiophilic adsorption, physiochemicalfractionation, or other antigen-specific affinity methods, for example,methods including protein A, G, and L antibody-binding ligands. Suchpurified antibodies may or may not have structural characteristics thatare different from human monoclonal antibodies that are not purified.For example, conformational epitope changes for human monoclonalantibodies may occur upon purification. Antibodies may be bound toadditional molecules that are removed upon purification. Accordingly,such purified antibodies may or may not have different functionalactivity. The anti-LAM and anti-PIM6/LAM antibodies of the presentinvention may have a number of structural modifications. For example,the anti-LAM and anti-PIM6/LAM antibodies of the present invention maybe glycosylated, PEGylated, or otherwise chemically modified in such amanner as to affect the stability, function, bioavailability, epitoperecognition, or other functional activity. The anti-LAM andanti-PIM6/LAM antibodies of the present invention may be engineeredvariants and/or derivatives of those antibodies described below, and mayor may not possess functional or structural equivalence. Accordingly,such variants and/or derivatives are still considered within the scopeof the present invention, so long as they are derived or engineered atleast in part from an isolated human monoclonal anti-LAM oranti-PIM6/LAM antibody, and/or recognize at least one epitope on LAM.

1. A194-01

In some embodiments, the present invention is directed to the humanmonoclonal antibody A194-01 including variants and/or derivativesthereof. A194-01 is specific for LAM. A194-01 possesses very highbinding activity for LAM, for example, the IgG isotype of A194-01 mayexhibit 50% maximal binding activity of the antibody at a concentrationof approximately 20 ng/ml, thus signifying a high affinity for LAM.A194-01 was originally isolated and purified as an IgG, however, A194-01may exist in a number of isotypes, as well as engineered and recombinantisotypes, including but not limited to IgG, IgA, IgM, monovalent singlechain Fv (scFv) fragments, Fab proteins, divalent scFv fragments, singlechain scFv fragments (monomers) wherein individual variable light andvariable heavy regions are joined by e.g. a flexible linker, and dimericscFv proteins in which two scFv monomers are joined to one another (FIG.1A) Some particular engineered variants and/or derivatives of A194-01include, but are not limited to the following. One engineered variantand/or derivative of A194-01 comprises a tetravalent scFv-IgG, formed byjoining the A194-01 scFv antigen to the N-terminus of A194-01 IgG (FIG.1B, FIG. 17 ), which may increase binding affinity and broaden the rangeof epitopes recognized (examples of this are given in FIGS. 14 and 15 ).The tetravalent scFv-IgG may comprise leader-VH-VL-IgG, or may compriseleader-VL-VH-IgG. One having ordinary skill in the art will appreciatethat engineered scFv-IgG variants and/or derivatives may have valencesbeyond just tetravalent. Another engineered variant and/or derivative ofA194-01 comprises a pentavalent IgM, generated by converting a dimericA194-01 IgG to a human IgM contain domain, wherein such pentavalent IgMcontains 10 binding sites (FIG. 1B). One of ordinary skill in the artwill appreciate that further combinations of A194-01 antigenic fragmentsare possible and are considered within the scope of this invention,particularly those antibody fragments that display complementaritydetermining regions (CDRs) specific to A194-01.

The IgG isotype of A194-01 recognizes a unique and complex epitope thatis expressed on unmodified Ara4 and Ara6 side-chains and on side-chainsbearing a single mannose. Although A194-01 does not recognize sidechains bearing di- or tri-mannose substitutions, it does react with suchstructures if they are further modified with an MSX substituent.Accordingly, the IgG isotype of A194-01 binds to PILAM and ManLAM withhigh affinity, and also binds strongly with uncapped versions of bothAra4 and Ara6 structures, and binds somewhat less strongly to singlemannose-capped and MSX-substituted Ara4/Ara6 structures, but poorly ifat all to di-substituted and tri-substituted ManLAM (FIG. 4 ). Withoutwishing to be bound by theory, the dramatically different effect ofattachment of mannose versus MSX to the terminal mannose of themono-mannosylated Ara4 structure may reflect a difference between theα(1→2) linkage of the mannose and α(1→4) linkage of the MSXsubstitution. Engineered variants and/or derivatives of A194-01,including those that possess higher valencies, may exhibit broaderepitope specificity than the A194-01 IgG isotype (FIG. 14 ), and mayfurther exhibit enhanced affinity for LAM (FIG. 15 ). For example, thetetravalent scFv-IgG engineered A194-01 and the engineered IgA and IgMisotypes bind to both Ara4 and Ara6 structures with higher affinity thanthe A194-01 IgG isotype, and furthermore, also recognize di-mannose andtri-mannose capped structures that the IgG isotype binds to poorly (FIG.14 ). Because pathogenic species of the Mycobacteriumtuberculosis-complex predominantly exhibit di-mannose capped structures,these engineered variants and/or derivatives of A194-01, includingscFv-IgG and IgM isotypes, may prove particularly useful for diagnostickits and methods, as well as for therapeutic use.

Further engineered variants and/or derivatives of A194-01 include thoseantibodies wherein the IgG1 Fc domain is converted to IgG3, which ismore opsogenic, or by generating multimeric versions, by substitutingthe IgG1 constant domain by dimeric IgA or pentameric or hexameric IgM.Without wishing to be bound by theory, this may significantly enhanceavidity of the anti-LAM antibodies by increasing the flexibility andrange of bivalent and multivalent binding, which contributes to affinity(FIG. 1 ). This is of potential clinical significance, as treatmentswould be particularly valuable in cases of exposure or infection withMDR or X-MDR strains of Mycobacterium tuberculosis, which cannot beeffectively treated with traditional antibiotics.

TABLE 1 A194-01 Complementarity Determining Regions (CDR) Light ChainCDR1- RSIRSA (SEQ ID NO: 1) CDR2- GAS (SEQ ID NO: 2)CDR3- QQYDFWYTF (SEQ ID NO: 3) Heavy Chain CDR1- GFNFEDFG (SEQ ID NO: 4)CDR2- ISWNGANI (SEQ ID NO: 5) CDR3- IDWYRDDYYKMDV (SEQ ID NO: 6)

One of ordinary skill in the art will appreciate that CDRs are crucialto the diversity of antigen specificities. One having ordinary skill inthe art will further appreciate that CDR3 is the most variable of CDRregions, and as such bears the greatest importance, with diversity inthe CDR3 region of the variable heavy chain being sufficient for mostantibody specificities. Accordingly, in some embodiments, the anti-LAMantibodies have a CDR1, CDR2, and CDR3 region of the variable lightchain as set forth in SEQ ID NOS: 1, 2 and 3, respectively. In someembodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3 regionof the variable light chain as set forth in SEQ ID NOS: 1, 2, and 3respectively with conservative sequence modifications. In someembodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3 regionof the variable light chain having up to 95% identity with SEQ ID NOS:1, 2, and 3 respectively. In other embodiments, the anti-LAM antibodieshave a CDR1, CDR2, and CDR3 region of the variable light chain having upto 90% identity with SEQ ID NOS: 1, 2, and 3 respectively. In otherembodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3 regionof the variable light chain having up to 85% identity with SEQ ID NOS:1, 2, and 3 respectively. In other embodiments, the anti-LAM antibodieshave a CDR1, CDR2, and CDR3 region of the variable light chain having upto 80% identity with SEQ ID NOS: 1, 2, and 3 respectively. In someembodiments, the anti-LAM antibodies have a CDR3 region of the variablelight chain as set forth in SEQ ID NO: 3. In some embodiments, theanti-LAM antibodies have a CDR3 region of the variable light chain asset forth in SEQ ID NO: 3 with conservative sequence modifications. Inother embodiments, the anti-LAM antibodies have a CDR3 region of thevariable light chain having up to 95% identity with SEQ ID NO: 3. Inother embodiments, the anti-LAM antibodies have a CDR3 region of thevariable light chain having up to 90% identity with SEQ ID NO: 3. Inother embodiments, the anti-LAM antibodies have a CDR3 region of thevariable light chain having up to 85% identity with SEQ ID NO: 3. Inother embodiments, the anti-LAM antibodies have a CDR3 region of thevariable light chain having up to 80% identity with SEQ ID NO: 3.

In some embodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3region of the variable heavy chain as set forth in SEQ ID NOS: 4, 5 and6, respectively. In some embodiments, the anti-LAM antibodies have aCDR1, CDR2, and CDR3 region of the variable heavy chain as set forth inSEQ ID NOS: 4, 5 and 6, respectively with conservative sequencemodifications. In some embodiments, the anti-LAM antibodies have a CDR1,CDR2, and CDR3 region of the variable heavy chain having up to 95%identity with SEQ ID NOS: 4, 5, and 6 respectively. In otherembodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3 regionof the variable heavy chain having up to 90% identity with SEQ ID NOS:4, 5, and 6 respectively. In other embodiments, the anti-LAM antibodieshave a CDR1, CDR2, and CDR3 region of the variable heavy chain having upto 85% identity with SEQ ID NOS: 4, 5, and 6 respectively. In otherembodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3 regionof the variable heavy chain having up to 80% identity with SEQ ID NOS:4, 5, and 6 respectively. In some embodiments, the anti-LAM antibodieshave a CDR3 region of the variable heavy chain as set forth in SEQ IDNO: 6. In some embodiments, the anti-LAM antibodies have a CDR3 regionof the variable heavy chain as set forth in SEQ ID NO: 6 withconservative sequence modifications. In other embodiments, the anti-LAMantibodies have a CDR3 region of the variable heavy chain having up to95% identity with SEQ ID NO: 6. In other embodiments, the anti-LAMantibodies have a CDR3 region of the variable heavy chain having up to90% identity with SEQ ID NO: 6. In other embodiments, the anti-LAMantibodies have a CDR3 region of the variable heavy chain having up to85% identity with SEQ ID NO: 6. In other embodiments, the anti-LAMantibodies have a CDR3 region of the variable heavy chain having up to80% identity with SEQ ID NO: 6.

In the experiments described herein, the A194-01 antibody was expressedby transfection of H and L chain vectors in Expi293 cells and culturedin standard Expi293 serum-free medium for several days. The secretedantibody was purified from the culture supernatant by affinitychromatography on columns conjugated with Protein A or Protein Gligands. The bound antibodies were released from the ligands bytreatment with low pH buffer (0.2 M glycine-HCl, pH 2.5) and neutralizedwith 1/50 volume of 2 M tris buffer buffer (pH 8.8). The buffer wasexchanged with PBS by dialysis or by several rounds of concentration oncentrifugal filters (Amicon Ultra centrifugal filters, 30K mw limit).

The amino acid (aa) and nucleic acid (nt) sequences for A194 heavy andlight chain sequences are as follows:

A194 Heavy chain nt sequence: (SEQ ID NO: 39)CAAGTGCAGCTGTTGGAGTCTGGGGGAGGTGTGGTACGGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCAACTTTGAAGATTTTGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGAGTGGGTCTCTAGTATTAGTTGGAATGGTGCTAATATAGGCTATGTAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTATATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCTTATATTACTGTGCGATAGACTGGTACAGAGACGACTACTACAAGATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGCCTCGACCAAGGGCCCATCGGTCTTCCCGCTAGCGCCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA A194 Heavy chain aa sequence:(SEQ ID NO: 40) QVQLLESGGGVVRPGGSLRLSCAASGFNFEDFGMSWVRQAPGKGLEWVSSISWNGANIGYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAIDWYRDDYYKMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK*A194 Light chain nt sequence (kappa): (SEQ ID NO: 41)GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTCTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCGGAGTATTCGCAGCGCCTTAGCCTGGTACCAGCACAAACCTGGCCAGGCTCCCAGGCTCCTCATCTTTGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCGTCAGCAGCATACGGTCTGAGGATTCTGCAGTTTATTACTGTCAGCAGTATGATTTCTGGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTCGACAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTAGA194 Light chain aa sequence (kappa): (SEQ ID NO: 42)EIVMTQSPATLSVSPGERATLSCRASRSIRSALAWYQHKPGQAPRLLIFGASTRATGIPARFSGSGSGTDFTLTVSSIRSEDSAVYYCQQYDFWYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC*2. P30B9

In some embodiments, the present invention is directed to therecombinant human monoclonal antibody P30B9 including variants and/orderivatives thereof. P30B9 is specific for LAM. P30B9 was originallyisolated and purified as an IgM, however, P30B9 may exist in a number ofisotypes, as well as engineered and recombinant isotypes, including butnot limited to IgM, IgG, IgA, as well as antigenic fragments thereof,including but not limited to monovalent single chain Fv (scFv)fragments, Fab proteins, divalent scFv fragments, single chain scFvfragments (monomers) wherein individual variable light and variableheavy regions are joined by e.g. a flexible linker, and dimeric scFvproteins in which two scFv monomers are joined to one another.

The IgM isotype of P30B9 binds most potently to di-mannose substitutedAra4 and Ara6 LAM epitopes with the Manp-α (1→2)-Manp-(1→5)-Arafstructures (FIGS. 4, 16 and 18 ) although other Manp-α substitutedstructures (e.g., structure 2, 4 and 59 in FIG. 8 ) may also berecognized with lower affinities. The preferential recognition of P30B9for di-mannose capped LAM has potential clinical relevance, sincedi-mannose caps are reported to be the dominant LAM modification onvirulent strains of the Mycobacterium tuberculosis-complex. Withoutwishing to be bound by theory, it is believed that terminal mannosylunits mediate binding of LAM from virulent strains of the Mycobacteriumtuberculosis-complex to human macrophage and other immune cells thatleads to the perturbation of immune function and establishment of stableinfection. Without wishing to be bound by theory, binding of the mannosecaps to the mannose receptor is believed to limit phagosome-lysosome(P-L) fusion and facilitate survival of the bacterium in infectedmacrophages. The specificity of P30B9 for di-mannose capped LAM isindicated by the specificity of this mAb for glyconjugates bearing thisstructure, and in the fact that the IgM isotype of P30B9 bindsspecifically to LAM derived from either Mycobacterium tuberculosis, butnot to LAM from Mycobacterium smegmatis or Mycobacterium leprae, whichdo not contain di-mannose capped LAM epitopes. This is in contrast tothe IgG isotype of A194-01, which binds to PILAM, uncapped Ara4/Ara6residues, and mono-mannose capped LAM epitopes, all of which are commonin Mycobacterium smegmatis and Mycobacterium leprae. Like the IgMisotype of P30B9, the IgM isotype of A194-01 is able to bind todi-mannose and tri-mannose capped LAM epitopes (FIG. 14 ), presumabledue to an increased binding avidity.

Therefore, the IgM isotypes of P30B9 may serve as an importantimmunodiagnostic reagent for detecting infection by virulent members ofthe Mycobacterium tuberculosis-complex and distinguishing said virulentmembers other nonpathogenic mycobacterial species, as it is specific todi-mannose capped LAM. Furthermore, the IgM isotype of the P30B9antibody as well as engineered variants and/or derivatives of A194-01may possess immunotherapeutic activity that limit infection andpathogenesis of virulent members of the Mycobacteriumtuberculosis-complex and may be suitable for use in therapy, either incombination with traditional antibiotics, additional antibodies, oralone, or may be used as a passive immunotherapeutic agent. The IgMisotype of P30B9 binds specifically to ManLAM derived from Mycobacteriumtuberculosis with high affinity (FIGS. 2A,B), but not to PILAM derivedfrom Mycobacterium smegmatis (FIG. 2C).

Engineered variants and/or derivatives of P30B9 may include, forexample, P30B9 expressed in the IgA isotype, including dimeric IgA1 andIgA2. Without wishing to be bound by theory, it is believed thatpolyvalency is required for P30B9 function, as this antibody wasisolated as an IgM, and is not active when expressed as an IgG. Thepresent invention shows that P30B9 is active in engineered IgA isotypes,including dimeric IgA1 and IgA2. This was tested by moving the P30B9 VHdomain into IgA1 and IgA2 vectors. IgA1 differs from IgA2 mostly by thepresence of a 16 amino-acid insertion, comprised of a repeat of 8 aminoacids rich in proline, serine, and threonine, and modified with 3-6,O-linked oligosaccharides [FIG. 5 ]. The binding activity of theengineered IgA forms of P30B9 to ManLAM were compared to those of theIgG and IgM forms. The IgM form had the highest activity, while both ofthe IgA forms were also able to bind to ManLAM, with the IgA2 formshowing weaker activity than the IgA1 form, and the IgG form wasinactive in an ELISA against ManLAM (FIG. 6 ).

TABLE 2 P30B9 Complementarity Determining Regions (CDR) Light ChainCDR1- QSINSN (SEQ ID NO: 7) CDR2- KAS (SEQ ID NO: 8)CDR3- QQYKAFKTF (SEQ ID NO: 9) Heavy ChainCDR1- GGSFSGYY (SEQ ID NO: 10) CDR2- FDLGGSITHSRGT (SEQ ID NO: 11)CDR3- RGLAMGGTKEFDS (SEQ ID NO: 12)

One of ordinary skill in the art will appreciate that CDRs are crucialto the diversity of antigen specificities. One having ordinary skill inthe art will further appreciate that CDR3 is the most variable of CDRregions, and as such bears the greatest importance, with diversity inthe CDR3 region of the variable heavy chain being sufficient for mostantibody specificities. Accordingly, in some embodiments, the anti-LAMantibodies have a CDR1, CDR2, and CDR3 region of the variable lightchain as set forth in SEQ ID NOS: 7, 8 and 9, respectively. In someembodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3 regionof the variable light chain as set forth in SEQ ID NOS: 7, 8 and 9,respectively with conservative sequence modifications. In someembodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3 regionof the variable light chain having up to 95% identity with SEQ ID NOS:7, 8, and 9 respectively. In other embodiments, the anti-LAM antibodieshave a CDR1, CDR2, and CDR3 region of the variable light chain having upto 90% identity with SEQ ID NOS: 7, 8, and 9 respectively. In otherembodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3 regionof the variable light chain having up to 85% identity with SEQ ID NOS:7, 8, and 9 respectively. In other embodiments, the anti-LAM antibodieshave a CDR1, CDR2, and CDR3 region of the variable light chain having upto 80% identity with SEQ ID NOS: 7, 8, and 9 respectively. In someembodiments, the anti-LAM antibodies have a CDR3 region of the variablelight chain as set forth in SEQ ID NO: 9. In some embodiments, theanti-LAM antibodies have a CDR3 region of the variable light chain asset forth in SEQ ID NO: 9 with conservative sequence modifications. Inother embodiments, the anti-LAM antibodies have a CDR3 region of thevariable light chain having up to 95% identity with SEQ ID NO: 9. Inother embodiments, the anti-LAM antibodies have a CDR3 region of thevariable light chain having up to 90% identity with SEQ ID NO: 9. Inother embodiments, the anti-LAM antibodies have a CDR3 region of thevariable light chain having up to 85% identity with SEQ ID NO: 9. Inother embodiments, the anti-LAM antibodies have a CDR3 region of thevariable light chain having up to 80% identity with SEQ ID NO: 9.

In some embodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3region of the variable heavy chain as set forth in SEQ ID NOS: 10, 11and 12, respectively. In some embodiments, the anti-LAM antibodies havea CDR1, CDR2, and CDR3 region of the variable heavy chain as set forthin SEQ ID NOS: 10, 11 and 12, respectively with conservative sequencemodifications. In some embodiments, the anti-LAM antibodies have a CDR1,CDR2, and CDR3 region of the variable heavy chain having up to 95%identity with SEQ ID NOS: 10, 11 and 12, respectively. In otherembodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3 regionof the variable heavy chain having up to 90% identity with SEQ ID NOS:10, 11 and 12, respectively. In other embodiments, the anti-LAMantibodies have a CDR1, CDR2, and CDR3 region of the variable heavychain having up to 85% identity with SEQ ID NOS: 10, 11 and 12,respectively. In other embodiments, the anti-LAM antibodies have a CDR1,CDR2, and CDR3 region of the variable heavy chain having up to 80%identity with SEQ ID NOS: 10, 11 and 12, respectively. In someembodiments, the anti-LAM antibodies have a CDR3 region of the variableheavy chain as set forth in SEQ ID NO: 12. In some embodiments, theanti-LAM antibodies have a CDR3 region of the variable heavy chain asset forth in SEQ ID NO: 12 with conservative sequence modifications. Inother embodiments, the anti-LAM antibodies have a CDR3 region of thevariable heavy chain having up to 95% identity with SEQ ID NO: 12. Inother embodiments, the anti-LAM antibodies have a CDR3 region of thevariable heavy chain having up to 90% identity with SEQ ID NO: 12. Inother embodiments, the anti-LAM antibodies have a CDR3 region of thevariable heavy chain having up to 85% identity with SEQ ID NO: 12. Inother embodiments, the anti-LAM antibodies have a CDR3 region of thevariable heavy chain having up to 80% identity with SEQ ID NO: 12.

In the experiments described herein, the P30B9 antibody was expressed bytransfection of H and L chain vectors in Expi293 cells and cultured instandard Expi293 serum-free medium for several days. The secretedantibody was purified from the culture supernatant by affinitychromatography on columns conjugated with Protein L ligand. The boundantibody was released from the ligands by treatment with low pH buffer(0.2 M glycine-HCl, pH 2.5) and neutralized with 1/50 volume of 2 M trisbuffer buffer (pH 8.8). The buffer was exchanged with PBS by dialysis orby several rounds of concentration on centrifugal filters (Amicon Ultracentrifugal filters, 30K mw limit).

The amino acid sequences for P30B9 heavy chain and light chain and theircomparison with its closest germline are shown in FIG. 22 . The aminoacid and nucleotide sequences for P30B9 including the CDR3 region arecopied below:

P30B9-Heavy chain variable region:  (SEQ ID NO: 43)QVQLQQWGAGLLKPSETLSLTCAVY GGSFSGYY WSWIRQSPETGLEWLGE FDLGGS ITHSRGTNYNPSLKSRVTISGDTSKNQFSLKLTSVTAADTAVYYC ARGLAMGGTKEFDSP30B9-Light chain variable region:  (SEQ ID NO: 44)DIQMTQSPDSLSASVGDRITITCRAS QSINSN LAWYQQKPGKAPKLLIY KASDLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYKAFKT P30B9-Heavy chain DNA sequence: (SEQ ID NO: 45)caggtgcagctacagcagtggggcgcaggactgttgaagccttcggagaccctgtccctcacctgcgctgtctatggtgggtccttcagtggttactactggagctggatccgccagtccccagagacggggctggagtggcttggcgaaTTCGATCTTGGTGGAAGCatcactcatagtagaggcaccaactacaacccgtcgctcaagagtcgagtcaccatctcaggagacacgtccaagaaccagttctccctgaaactgacctctgtgaccgccgcggacacggctgtctattactgtgcgagaggtttagcaatgggtggaactaaggagtttgactcctggggccagggaaccctggtcaccgtctcctcag  P30B9-Light chain: (SEQ ID NO: 46)gacatccagatgacccagtctccagactccctgtctgcatctgtaggagacagaatcaccatcacttgccgggccagtcagagtattaatagtaatttggcctggtatcagcagaaaccggggaaagcccctaagctcctgatctataaggcgtctgatttagaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttattattgccaacagtataaagcattcaagacgttcggccacgggaccaaggtggaaatcaaac3. P95C1

In some embodiments, the present invention is directed to therecombinant human monoclonal antibody P95C1 including variants and/orderivatives thereof. P95C1 is specific for an epitope shared by LAM, LMand PIM6. Although P95C1 was originally isolated and purified as an IgM,P95C1 is also active when expressed in other isotypes, including but notlimited to IgG and IgA forms.

One of ordinary skill in the art will appreciate that CDRs are crucialto the diversity of antigen specificities. One having ordinary skill inthe art will further appreciate that CDR3 is the most variable of CDRregions, and as such bears the greatest importance, with diversity inthe CDR3 region of the variable heavy chain being sufficient for mostantibody specificities. Accordingly, in some embodiments, the anti-LAMantibodies have a CDR1, CDR2, and CDR3 region of the variable lightchain as set forth in SEQ ID NOS: 13, 14 and 15, respectively. In someembodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3 regionof the variable light chain as set forth in SEQ ID NOS: 13, 14 and 15,respectively with conservative sequence modifications. In someembodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3 regionof the variable light chain having up to 95% identity with SEQ ID NOS:13, 14 and 15, respectively. In other embodiments, the anti-LAMantibodies have a CDR1, CDR2, and CDR3 region of the variable lightchain having up to 90% identity with SEQ ID NOS: 13, 14 and 15,respectively. In other embodiments, the anti-LAM antibodies have a CDR1,CDR2, and CDR3 region of the variable light chain having up to 85%identity with SEQ ID NOS: 13, 14 and 15, respectively. In otherembodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3 regionof the variable light chain having up to 80% identity with SEQ ID NOS:13, 14 and 15, respectively. In some embodiments, the anti-LAMantibodies have a CDR3 region of the variable light chain as set forthin SEQ ID NO: 15. In some embodiments, the anti-LAM antibodies have aCDR3 region of the variable light chain as set forth in SEQ ID NO: 15with conservative sequence modifications. In other embodiments, theanti-LAM antibodies have a CDR3 region of the variable light chainhaving up to 95% identity with SEQ ID NO: 15. In other embodiments, theanti-LAM antibodies have a CDR3 region of the variable light chainhaving up to 90% identity with SEQ ID NO: 15. In other embodiments, theanti-LAM antibodies have a CDR3 region of the variable light chainhaving up to 85% identity with SEQ ID NO: 15. In other embodiments, theanti-LAM antibodies have a CDR3 region of the variable light chainhaving up to 80% identity with SEQ ID NO: 15.

In some embodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3region of the variable heavy chain as set forth in SEQ ID NOS: 16, 17and 18, respectively. In some embodiments, the anti-LAM antibodies havea CDR1, CDR2, and CDR3 region of the variable heavy chain as set forthin SEQ ID NOS: 16, 17 and 18, respectively with conservative sequencemodifications. In some embodiments, the anti-LAM antibodies have a CDR1,CDR2, and CDR3 region of the variable heavy chain having up to 95%identity with SEQ ID NOS: 16, 17 and 18, respectively. In otherembodiments, the anti-LAM antibodies have a CDR1, CDR2, and CDR3 regionof the variable heavy chain having up to 90% identity with SEQ ID NOS:16, 17 and 18, respectively. In other embodiments, the anti-LAMantibodies have a CDR1, CDR2, and CDR3 region of the variable heavychain having up to 85% identity with SEQ ID NOS: 16, 17 and 18,respectively. In other embodiments, the anti-LAM antibodies have a CDR1,CDR2, and CDR3 region of the variable heavy chain having up to 80%identity with SEQ ID NOS: 16, 17 and 18, respectively. In someembodiments, the anti-LAM antibodies have a CDR3 region of the variableheavy chain as set forth in SEQ ID NO: 18. In some embodiments, theanti-LAM antibodies have a CDR3 region of the variable heavy chain asset forth in SEQ ID NO: 18 with conservative sequence modifications. Inother embodiments, the anti-LAM antibodies have a CDR3 region of thevariable heavy chain having up to 95% identity with SEQ ID NO: 18. Inother embodiments, the anti-LAM antibodies have a CDR3 region of thevariable heavy chain having up to 90% identity with SEQ ID NO: 18. Inother embodiments, the anti-LAM antibodies have a CDR3 region of thevariable heavy chain having up to 85% identity with SEQ ID NO: 18. Inother embodiments, the anti-LAM antibodies have a CDR3 region of thevariable heavy chain having up to 80% identity with SEQ ID NO: 18.

TABLE 3 P95C1 Complimentary Determining Regions (CDR) Light ChainCDR1: QNVLDSANNRNY (SEQ ID NO: 13) CDR2: WAS (SEQ ID NO: 14)CDR3: TQYHRLPHT (SEQ ID NO: 15) Heavy ChainCDR1: GGSINTNNW (SEQ ID NO: 16) CDR2: IHRHGDT (SEQ ID NO: 17)CDR3: CPLGYCSGDDCHRVA (SEQ ID NO: 18)

The P95C1 IgM/κantibody was originally identified in supernatants ofBCL6/Bcl-xL transduced memory B cells and cloned from these cells intoIgM/κexpression vectors using the standard RT-PCR protocol. The antibodywas expressed by transfection of H and L chain vectors in Expi293 cellsand cultured in standard Expi293 serum-free medium for several days. Thesecreted antibody was purified from the culture supernatant by affinitychromatography on columns conjugated with Protein L ligand. The boundantibody was released from the ligands by treatment with low pH buffer(0.2 M glycine-HCl, pH 2.5) and neutralized with 1/50 volume of 2 M trisbuffer buffer (pH 8.8). The buffer was exchanged with PBS by dialysis orby several rounds of concentration on centrifugal filters (Amicon Ultracentrifugal filters, 30K mw limit).

The amino acid sequences for P95C1 heavy chain and light chain and theircomparison with its closest germline are shown in FIG. 23 . The aminoacid and nucleotide sequences for P95C1 including the CDR3 region arecopied below:

P95C1-Heavy chain variable region: (SEQ ID NO: 47)EVQLLESGPGLVRPWGTLSLTCAVS GGSINTNNW WSWVRQSPGKGLEWIGE IHRHGDTNYNPSLKRRVSISMDESMNQFSLRLISVTAADTAVYYC CPLGYCSGDDCHRVAP95C1-Light chain variable region: (SEQ ID NO: 48)DIQMTQSPSSLSVSLGERATINCKSS QNVLDSANNRNY FGWYQQKPGQPPKLLIS WASTRESGVPDRFSGSGSGTDFTLIISGLQVEDVAVYYC TQYHRLPHT P95C1-Heavy chain:(SEQ ID NO: 49) gaggtgcagctcttggagtcgggcccaggactggtgaggccttgggggactctgtccctcacctgcgctgtctctggtggctccatcaatactaataactggtggagttgggtccgccagtccccggggaaggggctggagtggattggagaaatccatcgtcatggggacaccaactacaacccgtcactcaagaggcgagtctccatatcgatggacgagtccatgaaccagttctccctgaggcttatctctgtgaccgccgcggacacggccgtgtattactgttgtcccctaggatattgtagtggtgatgactgtcaccgagttgcctggggccggggaatcctggtcaccgtctcttcag P95C1-Light chain: (SEQ ID NO: 50)gacatccagatgacccagtctccatcctccctgtctgtgtctctgggcgagagggccaccatcaactgcaagtccagccagaatgttttagacagcgccaacaataggaactacttcggttggtaccagcagaaaccagggcagcctcctaagctgctcatttcctgggcatctacacgggaatccggggtccctgaccgattcagtggcagcggctctgggacagacttcactctcatcatcagcggcctgcaggttgaagatgtggcagtttattactgtacacagtatcatagacttcctcacaccttcggccaagggacacgactggaaattaaac

E. Further Variants and/or Derivatives

One of ordinary skill in the art will appreciate that given the CDRregions of A194-01, P30B9, and P95C1, a wide number of engineeredvariants and/or derivatives of the anti-LAM antibodies disclosed hereinmay be constructed. For example, the anti-LAM antibodies of the presentinvention may be engineered into chimeric antibodies, humanizedantibodies, and chimeric/humanized antibodies that exhibit affinity toone or more LAM epitopes. The antibodies may be engineered intobispecific antibodies, or may be engineered such that a single antibodyconstruct binds to multiple LAM epitopes.

As described herein, the anti-LAM antibodies of the present inventionmay be engineered as homologous scFv-IgG constructs or as heterologousscFv-IgG constructs. Homolgous scFv-IgG constructs of A194-01 aredetailed in this Application (FIG. 17A). One non-limiting example of aheterologous scFv-IgG construct would be where the VH and VL chains ofP30B9 were joined to the A194-01 IgG by a linker (FIG. 17B), althoughother VH/VL chains could be used, for example other anti-LAM antibodiessuch as murine anti-LAM antibodies. This may allow recognition ofdistinct epitopes in a single antigen molecule and may enhancemultivalent binding and lead to increased affinity. Alternatively,heterologous scFv-IgG constructs may generate bispecific antibodies ifthe additional VH/VL chains target an antigen other than LAM.

The anti-LAM antibodies of the present invention may also be engineeredto create scFv-IgM constructs, including both homologous andheterologous scFv-IgM constructs. A non-limiting example of a homologousscFv-IgM would be where P30B9 VH/VL chains are joined to the P30B9 IgM.In this construct, all binding sites would possess the same epitopespecificity. A non-limiting example of a heterologous scFv-IgM constructis where the A194-01 scFv is joined to the P30B9 IgM, as opposed to theIgG constant domain [non-limiting [FIG. 17C]. Such engineered variantand/or derivative construct would retain the IgM-dependent recognitionof dimannose epitopes of the parental P30B9 mAb and add the additionalbinding specificity of the A194-01 scFv. This may allow recognition ofunique epitope arrays and lead to enhanced affinities, which could bevaluable for improved point-of-care antigen detection assays.

F. Diagnostic Kits and Methods

One embodiment of the present invention relates to diagnostic kits andmethods for the detection and/or quantification of LAM and/or PIM6 in asample. As described herein, the anti-LAM antibodies A194-01 and P30B9,as well as the anti-PIM6/LAM antibody P95C1, including engineeredvariants and/or derivatives thereof, may be effective in detectingand/or quantifying the amount of LAM and/or PIM6 present in a sample.The LAM or PIM6 may be derived from any source, such as fromMycobacterium tuberculosis or Mycobacterium smegmatis, or from a serumor urine sample from a patient, e.g. a patient infected with a virulentstrain of the Mycobacterium tuberculosis-complex. The LAM may be e.g.PILAM, ManLAM, or uncapped/unmodified AraLAM from other mycobacterialstrains, such as M. leprae. These strains differ in the nature andextent of capping that occurs, and different antibody combinations wouldtherefore have different specificities for the different forms, allowingsome level of differentiation or typing to be performed. In particular,the IgM and engineered IgA1 isotype of P30B9, as well as the engineeredIgM and scFv-IgG isotypes of A194-01, would be well-suited for detectingand/or quantifying di-mannose substituted ManLAM in a sample from a TBpatient, which in some circumstances may comprise 80% of said LAM, andvarious isotypes of P30B9 would be especially effective at detectingand/or quantifying LAM bearing di-mannose substituted Ara6 residues,which as described herein are particularly prevalent on LAM derived fromMycobacterium tuberculosis. Since the P95C1 epitope is highly conservedin all species of LAM, this antibody, when coupled with a secondantibody with the proper specificity, would be well-suited for detectingand/or quantifying various types of LAM in a sample. The IgG isotype ofA194-01 binds very effectively to various forms of LAM, especiallyunsubstituted LAM, mono-mannonsylated LAM, and PILAM, and so would beeffective at detecting and/or quantifying LAM derived from variousstrains of mycobacteria. The engineered IgM and scFv-IgG isotypes wouldalso be quite effective at detecting and/or quantifying the amount ofunsubstituted LAM, mono-mannonsylated LAM, and PILAM, and additionallymay bind to di- and tri-mannose substituted LAM. This endows theengineered variants and/or derivatives of A194-01 with greater epitoperecognition than the IgG isotype of A194-01 or the IgM isotype of P30B9,but at the expense of specificity for only those LAM epitopes that arespecific to virulent strains of Mycobacterium tuberculosis. In someembodiments, quantifying said specificity for LAM and/or PIM6 isachieved by comparing the signal intensity of a serially diluted controlsample having a known concentration of LAM and/or PIM6 in various directbinding assays or antigen-capture assays.

Because the IgG isotype of A194-01, the IgM/IgA isotypes of P30B9, andthe various isotypes of P95C1 bind to different LAM epitopes that arevariably expressed in different strains of Mycobacterium tuberculosis,these particular isotypes could be used to differentiate the originationof a source of LAM; di-mannose substituted LAM, in particular di-mannosesubstituted Ara6 residues comprise the majority of LAM residues invirulent strains of Mycobacterium tuberculosis, whereas unsubstitutedLAM/PILAM residues comprise the majority of LAM residues in fast growingnon-virulent strains such as Mycobacterium smegmatis. For example,samples comprising LAM that bind only to A194-01 IgG and not P30B9 IgMlikely did not originate from a virulent strain of Mycobacteriumtuberculosis, whereas samples that bind to both P30B0 IgM and A194-01IgG likely did originate from a virulent strain of Mycobacteriumtuberculosis or a species of mycobacteria that introduces a similarcapping motif.

Because the IgM/IgA isotypes of P30B9 are specific for di-mannosesubstituted ManLAM, which as detailed herein is the dominant form invirulent strains of Mycobacterium tuberculosis, said isotypes of P30B9are ideal candidates for diagnostic kits and methods of use fordiagnosing a patient as being infected with a virulent strain of theMycobacterium tuberculosis-complex. Furthermore, the engineered IgM andscFv-IgG variants and/or derivatives of A194-01 may be suitable for sucha use as they also recognize di-mannose and tri-mannose substitutedManLAM epitopes. Such a patient could have an ongoing or activeinfection, or the infection could be latent. The strain could bemulti-drug resistant (MDR) or could be extensively-drug resistant (XDR).Specifically regarding patients having latent infections, change in LAMconcentration in the serum or urine may be of particular importance, asincreases in concentrations may signify a change to active infection.Alternatively, a decrease in concentration in an individual who has anactive infection may signify that treatment is effective and should becontinued, or an increase in concentration during treatment may indicatethat the current treatment is not effective and should be eliminated,changed and/or modified.

The methods for diagnosing infection may including contacting abiological sample from said patient, e.g. blood, plasma, urine, sputum,or other bodily fluid, with at least one anti-LAM antibody and/or atleast one anti-PIM6/LAM antibody of the present invention, particularlythose anti-LAM antibodies that recognize di-mannose substituted ManLAMand those anti-PIM6/LAM antibodies that recognize at least onepolymannose structure in the PIM6 mannan domain. These include, forexample, IgM and IgA isotypes of P30B9, the engineered IgA, IgM andscFv-IgG isotypes of A194-01, and the various isotypes (IgG, IgM, IgA)of P95C1.

The antibodies used as the detecting reagent may be bound to reportermolecules such as those known in the art. The antibodies may be part ofa kit, e.g. bound to a substrate or part of a sandwich assay. The kitsmay include a first anti-LAM or anti-PIM6/LAM capture antibody, a secondanti-LAM or anti-PIM6/LAM detector (detection) antibody which is boundto a reporter molecule, and a support to which the capture anti-LAM oranti-PIM6/LAM antibody is bound. The first and second anti-LAM antibodymay bind to the same LAM epitopes which are present in multiple copieson a single LAM molecule, or preferably they may bind to differentepitopes present on a single LAM molecule. The LAM and PIM6 epitopes maybe any of those described herein. The kits may include a third captureor detector (detection) antibody which binds to a non-competing site ofthe first and second antibody. This may increase the number of moleculescaptured and number of detector molecules bound and the strength of thecorresponding signal.

The kits may include instructions for use, and may further containvarious reagents, solvents, diluents, and/or pharmaceutically acceptablepreservatives. The sensitivity of different biotin-labeled anti-LAMmonoclonal antibodies in such an assay was conducted [FIG. 7 ]. In thisassay, the murine anti-LAM antibody CS-35 was used to capture ManLAMfrom solution. This antibody was selected because of its broadspecificity. CS-35 (250 ng/well) was used to capture ManLAM fromsolutions containing differing concentrations, and differentbiotinylated monoclonal antibodies were then used to probe for thepresence of ManLAM in the capture well. Using a cut-off of 3×SD ofbackground, the most sensitive probe was A194-01 IgM, which gave astrong signal (1.8 OD) for the highest dilution of ManLAM (0.016ng/well). This was superior to the two FIND murine antibodies, whichhave been previously considered to be the best available probes for thistype of assay.

G. Therapeutic Compositions, Methods, Vaccines, and Vectors

One embodiment of the present invention is directed towardspharmaceutical compositions comprising at least one anti-LAM antibody oranti-PIM6/LAM antibody of the present invention, as well as theirmethods of use in treating a patient in need thereof. The patient mayhave a latent or active infection by a virulent strain of Mycobacteriumtuberculosis, and of particular utility, the strain may be multi-drugresistant (MDR) or extensively drug resistant (XDR) to traditionaltherapies/antibiotics. The anti-LAM and anti-PIM6/LAM antibodiesutilized in these compositions and methods may be any anti-LAM antibodyor anti-PIM6/LAM antibody of the present invention, but of particularutility may be those anti-LAM antibodies that recognize di-mannosecapped ManLAM, particularly di-mannose capped Ara6 residues, e.g. P30B9IgM or IgA1/IgA2 isotype and pentavalent A194-01 IgM or tetravalentscFv-IgG isotype and various isotypes of P95C1.

A pharmaceutically acceptable anti-LAM antibody and/or anti-PIM6/LAMantibody composition suitable for patient administration will contain aneffective amount of the anti-LAM or anti-PIM6/LAM antibody or antibodiesin a formulation which both retains biological activity while alsopromoting maximal stability during storage within an acceptabletemperature range. The pharmaceutical compositions can also include,depending on the formulation desired, pharmaceutically acceptablediluents, pharmaceutically acceptable carriers and/or pharmaceuticallyacceptable excipients, or any such vehicle commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. The amount of an excipient that is usefulin the pharmaceutical composition or formulation of this invention is anamount that serves to uniformly distribute the antibody throughout thecomposition so that it can be uniformly dispersed when it is to bedelivered to a subject in need thereof. It may serve to dilute theantibody to a concentration which provides the desired beneficialpalliative or curative results while at the same time minimizing anyadverse side effects that might occur from too high a concentration. Itmay also have a preservative effect. Thus, for the antibody having ahigh physiological activity, more of the excipient will be employed. Onthe other hand, for any active ingredient(s) that exhibit a lowerphysiological activity, a lesser quantity of the excipient will beemployed.

The pharmaceutically acceptable anti-LAM antibody and/or anti-PIM6/LAMantibody composition may be in liquid form or solid form. A solidformulation is generally lyophilized and brought into solution prior toadministration for either single or multiple dosing. The formulationsshould not be exposed to extreme temperature or pH so as to avoidthermal denaturation. Thus, it is essential to formulate an antibodycomposition of the present invention within a biologically relevant pHrange. A solution buffered to maintain a proper pH range during storageis indicated, especially for liquid formulations stored for longerperiods of time between formulation and administration. To date, bothliquid and solid formulations require storage at lower temperatures(usually 2-8° C.) in order to retain stability for longer periods.Formulated antibody compositions, especially liquid formulations, maycontain a bacteriostat to prevent or minimize proteolysis duringstorage, including but not limited to effective concentrations (usually<1% w/v) of benzyl alcohol, phenol, m-cresol, chlorobutanol,methylparaben, and/or propylparaben. A bacteriostat may becontraindicated for some patients. Therefore, a lyophilized formulationmay be reconstituted in a solution either containing or not containingsuch a component. Additional components may be added to either abuffered liquid or solid antibody formulation, including but not limitedto sugars as a cryoprotectant (including but not necessarily limited topolyhydroxy hydrocarbons such as sorbitol, mannitol, glycerol anddulcitol and/or disaccharides such as sucrose, lactose, maltose ortrehalose) and, in some instances, a relevant salt (including but notlimited to NaCl, KCl or LiCl). Such antibody formulations, especiallyliquid formulations slated for long term storage, will rely on a usefulrange of total osmolarity to both promote long term stability attemperature of 2-8° C., or higher, while also making the formulationuseful for parenteral injection. An effective range of total osmolarity(the total number of molecules in solution) is from about 200 mOs/L toabout 800 mOs/L. It will be apparent that the amount of acyroprotectant, such as sucrose or sorbitol, will depend upon the amountof salt in the formulation in order for the total osmolarity of thesolution to remain within an appropriate range. Therefore a salt freeformulation may contain from about 5% to about 25% sucrose, with apreferred range of sucrose from about 7% to about 15%, with anespecially preferred sucrose concentration in a salt free formulationbeing from 10% to 12%. Alternatively, a salt free sorbitol-basedformulation may contain sorbitol within a range from about 3% to about12%, with a preferred range from about 4% to 7%, and an especiallypreferred range is from about 5% to about 6% sorbitol in a salt-freeformulation. Salt-free formulations will of course warrant increasedranges of the respective cryoprotectant in order to maintain effectiveosmolarity levels. These formulation may also contain a divalent cation(including but not necessarily limited to MgCl2, CaCl2) and MnCl2); anda non-32 ionic surfactant (including but not necessarily limited toPolysorbate-80 (Tween 80®), Polysorbate-60 (Tween 60®), Polysorbate-40(Tween 40®) and Polysorbate-20 (Tween 20®), polyoxyethylene alkylethers, including but not limited to Brij 58®, Brij 35®, as well asothers such as Triton X-100®, Triton X 114®, NP40®, Span 85 and thePluronic series of non-ionic surfactants (e.g., Pluronic 121)). Anycombination of such components, including probable inclusion of abacteriostat, may be useful to fill the antibody-containing formulationsof the present invention. The antibody composition of the presentinvention may also be a “chemical derivative”, which describes anantibody that contains additional chemical moieties which are notnormally a part of the immunogloblulin molecule (e.g., pegylation). Suchmoieties may improve the solubility, half-life, absorption, etc. of thebase molecule. Alternatively, the moieties may attenuate undesirableside effects of the base molecule or decrease the toxicity of the basemolecule.

Specific embodiments include PLGA microspheres, as discussed herein andas further known in the art, as well as polymer-based non-degradablevehicles comprising poly (ethylene-co-vinyl acetate; PEVAc).Additionally, controlled-release and localized delivery ofantibody-based therapeutic products is reviewed in Grainger, et al.,2004, Expert Opin. Biol. Ther. 4(7): 1029-1044), hereby incorporated byreference in its entirety. Suitable microcapsules capable ofencapsulating the antibody may also include hydroxymethylcellulose orgelatin-microcapsules and polymethyl methacrylate microcapsules preparedby coacervation techniques or by interfacial polymerization. See PCTpublication WO 99/24061 entitled “Method for Producing IGF-1Sustained-Release Formulations,” wherein a protein is encapsulated inPLGA microspheres, this reference which is hereby incorporated herein byreference in its entirety. In addition, microemulsions or colloidal drugdelivery systems such as liposomes and albumin microspheres, may also beused. Other preferred sustained-release compositions employ abioadhesive to retain the antibody at the site of administration. Asnoted above, the sustained-release formulation may comprise abiodegradable polymer into which the antibody is disposed, which mayprovide for non-immediate release. Non-injectable devices may bedescribed herein as an “implant”, “pharmaceutical depot implant”, “depotimplant”, “non-injectable depot” or some such similar term. Common depotimplants may include, but are not limited to, solid biodegradable andnon-biodegradable polymer devices (such as an extended polymer orcoaxial rod shaped device), as well as numerous pump systems also knownin the art. Injectable devices are split into bolus injections (releaseand dissipation of the drug subsequent to injection), and repository ordepot injections, which provide a storage reservoir at the site ofinjection, allowing for sustained-release of the biological agent overtime. A depot implant may be surgically tethered to the point ofdelivery so as to provide an adequate reservoir for the prolongedrelease of the antibody over time. Such a device will be capable ofcarrying the drug formulation in such quantities as therapeutically orprophylactically required for treatment over the pre-selected period.The depot implant may also provide protection to the formulation fromdegradation by body processes (such as proteases) for the duration oftreatment. As known in the art, the term “sustained-release” refers tothe gradual (continuous or discontinuous) release of such an agent fromthe block polymer matrix over an extended period of time. Regardless ofthe specific device, the sustained-release of the anti-LAM antibodyand/or anti-PIM6/LAM antibody composition will result in a localbiologically effective concentrations of the antibody. A sustainedrelease of the biological agent(s) will be for a period of a single day,several days, a week or more; but most likely for a month or more, or upto about six months, depending on the formulation. Natural or syntheticpolymers known in the art will be useful as a depot implant due tocharacteristics such as versatile degradation kinetics, safety, andbiocompatibility. These copolymers can be manipulated to modify thepharmacokinetics of the active ingredient, shield the agent fromenzymatic attack, as well as degrading over time at the site ofattachment or injection. The artisan will understand that there areample teachings in the art to manipulate the properties of thesecopolymers, including the respective production process, catalysts used,and final molecular weight of the sustained-release depot implant ordepot injection. Natural polymers include but are not limited toproteins (e.g., collagen, albumin or gelatin); polysaccharides(cellulose, starch, alginates, chitin, chitosan, cyclodextrin, dextran,hyaluronic acid) and lipids. Biodegradable synthetic polymers mayinclude but are not limited to various polyesters, copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983,Biopolymers 22:547-556), polylactides ([PLA]; U.S. Pat. No. 3,773,919and EP 058,481), polylactate polyglycolate (PLGA) such aspolylactide-co-glycolide (see, for example, U.S. Pat. Nos. 4,767,628 and5,654,008), polyglycolide (PG), polyethylene glycol (PEG) conjugates ofpoly(α-hydroxy acids), polyorthoesters, polyaspirins, polyphosphagenes,vinylpyrrolidone, polyvinyl alcohol (PVA), PVA-g-PLGA, PEGT-PBTcopolymer (polyactive), methacrylates, poly(N-isopropylacrylamide),PEO-PPO-PEO (pluronics), PEO-PPO-PAA copolymers, PLGA-PEO-PLGA,polyorthoesters (POE), or any combinations thereof, as described above(see, for example, U.S. Pat. No. 6,991,654 and U.S. Pat. Appl. No.20050187631, each of which is incorporated herein by reference in itsentirety, hydrogels (see, for example, Langer et al., 1981, J. Biomed.Mater. Res. 15:167-277; Langer, 1982, Chem. Tech. 12:98-105,non-degradable ethylene-vinyl acetate (e.g. ethylene vinyl acetate disksand poly(ethylene-co-vinyl acetate)), degradable lactic acid-glycolicacid copolymers such as the Lupron Depot™, poly-D-(−)-3-hydroxybutyricacid (EP 133,988), hyaluronic acid gels (see, for example, U.S. Pat. No.4,636,524), alginic acid suspensions, polyorthoesters (POE), and thelike. Polylactide (PLA) and its copolymers with glycolide (PLGA) havebeen well known in the art since the commercialization of the LupronDepot™, approved in 1989 as the first parenteral sustained-releaseformulation utilizing PLA polymers. Additional examples of productswhich utilize PLA and PLGA as excipients to achieve sustained-release ofthe active ingredient include Amidox (PLA; periodontal disease),Nutropin Depot (PLGA; with hGH), and the Trelstar Depot (PLGA; prostatecancer). Other synthetic polymers included but are not limited topoly(c-caprolactone), poly3-hydroxybutyrate, poly(β-malic acid) andpoly(dioxanone)]; polyanhydrides, polyurethane (see WO 2005/013936),polyamides, cyclodestrans, polyorthoesters, n-vinyl alcohol,polyethylene oxide/polyethylene terephthalate, polyphosphate,polyphosphonate, polyorthoester, polycyanoacrylate, polyethylenegylcol,polydihydropyran, and polyacytal. Non-biodegradable devices include butare not limited to various cellulose derivatives (carboxymethylcellulose, cellulose acetate, cellulose acetate propionate, ethylcellulose, hydroxypropyl methyl cellulose) silicon-based implants(polydimethylsiloxane), acrylic polymers, (polymethacrylate,polymethylmethacrylate, polyhydroxy(ethylmethylacrylate), as well aspolyethylene-co-(vinyl acetate), poloxamer, polyvinylpyrrolidone,poloxamine, polypropylene, polyamide, polyacetal, polyester, polyethylene-chlorotrifluoroethylene, polytetrafluoroethylene (PTFE or“Teflon™”), styrene butadiene rubber, polyethylene, polypropylene,polyphenylene oxide-polystyrene, poly-a-chloro-p-xylene,polymethylpentene, polysulfone and other related biostable polymers.Carriers suitable for sustained-release depot formulations include, butare not limited to, microspheres, films, capsules, particles, gels,coatings, matrices, wafers, pills or other pharmaceutical deliverycompositions. Examples of such sustained-release formulations aredescribed above. See also U.S. Pat. Nos. 6,953,593; 6,946,146;6,656,508; 6,541,033; and 6,451,346, the contents of each which areincorporated herein by reference. The dosage form must be capable ofcarrying the drug formulation in such quantities and concentration astherapeutically required for treatment over the pre-selected period, andmust provide sufficient protection to the formulation from degradationby body processes for the duration of treatment. For example, the dosageform can be surrounded by an exterior made of a material that hasproperties to protect against degradation from metabolic processes andthe risk of, e.g., leakage, cracking, breakage, or distortion. This canprevent expelling of the dosage form contents in an uncontrolled mannerunder stresses it would be subjected to during use, e.g., due tophysical forces exerted upon the drug release device as a result ofnormal joint articulation and other movements by the subject or forexample, in convective drug delivery devices, physical forces associatedwith pressure generated within the reservoir. The drug reservoir orother means for holding or containing the drug must also be of suchmaterial as to avoid unintended reactions with the active agentformulation, and is preferably biocompatible (e.g., where the dosageform is implanted, it is substantially non-reactive with respect to asubject's body or body fluids). Generally, the respective biologicalagent(s) is administered to an individual for at least 12 hours to atleast a week, and most likely via an implant designed to deliver a drugfor at least 10, 20, 30, 100 days or at least 4 months, or at least 6months or more, as required. The anti-LAM antibody and/or anti-PIM6/LAMantibody can be delivered at such relatively low volume rates, e.g.,from about 0.001 ml/day to 1 ml/day so as to minimize tissue disturbanceor trauma near the site where the formulation is released. Theformulation may be released at a rate of, depending on the specificbiological agent(s), at a low dose, e.g., from about 0.01 μg/hr or 0.1g/hr, 0.25 μg/hr, 1 μg/hr, generally up to about 200 μg/hr, or theformulation is delivered at a low volume rate e.g., a volume rate offrom about 0.001 ml/day to about 1 ml/day, for example, 0.01 microgramsper day up to about 20 milligrams per day. Dosage depends on a number offactors such as potency, bioavailability, and toxicity of the activeingredient (e.g., IgG antibody) used and the requirements of thesubject.

For in vivo treatment of human and non-human patients, the patient isadministered or provided a pharmaceutical formulation including at leastone anti-LAM antibody and/or at least one anti-PIM6/LAM antibody of thepresent invention. When used for in vivo therapy, the anti-LAM oranti-PIM6/LAM antibodies of the invention are administered to thepatient in therapeutically effective amounts (i.e., amounts thateliminate or reduce the total bacterial load). The antibodies areadministered to a human patient, in accord with known methods, such asintravenous administration, for example, as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes. The antibodies can beadministered parenterally, when possible, at the target cell site, orintravenously. In some embodiments, antibody is administered byintravenous or subcutaneous administration. Therapeutic compositions ofthe invention may be administered to a patient or subject systemically,parenterally, or locally. The above parameters for assessing successfultreatment and improvement in the disease are readily measurable byroutine procedures familiar to a physician.

For parenteral administration, the anti-LAM and anti-PIM6/LAM antibodiesmay be formulated in a unit dosage injectable form (solution,suspension, emulsion) in association with a pharmaceutically acceptable,parenteral vehicle. Examples of such vehicles include, but are notlimited, water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Non-aqueous vehicles include, but are not limitedto, fixed oils and ethyl oleate. Liposomes can be used as carriers. Thevehicle may contain minor amounts of additives such as substances thatenhance isotonicity and chemical stability, such as, for example,buffers and preservatives.

The anti-LAM and anti-PIM6/LAM antibodies of the present invention maybe administered to the host in any manner, strategy and/or combinationavailable in the art in amounts sufficient to offer a therapeutictreatment against infection by a virulent strain of Mycobacteriumtuberculosis-complex. These compositions may be provided to theindividual by a variety of routes known in the art, especiallyparenteral routes, including but in no way limited to parenteral routessuch as intravenous (IV), intramuscular (IM); or subcutaneous (SC)administration, with IV administration being the norm within the art oftherapeutic antibody administration. These compositions may beadministered as separate or multiple doses (i.e., administration of theantibody at staggered times by maintaining the sterile condition of theformulation through the treatment regime).

The dose and dosage regimen depends upon a variety of factors readilydetermined by a physician, such as the nature of the infection, forexample, its therapeutic index, the patient, and the patient's history.Generally, a therapeutically effective amount of an antibody isadministered to a patient. In some embodiments, the amount of antibodyadministered is in the range of about 0.01 mg/kg to about 1000 mg/kg ofpatient body weight, and any range in between. Depending on the type andseverity of the infection, about 0.1 mg/kg to about 50 mg/kg body weight(for example, about 0.1-15 mg/kg/dose) of antibody is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. The progress of this therapy is readily monitored byconventional methods and assays and based on criteria known to thephysician or other persons of skill in the art. The above parameters forassessing successful treatment and improvement in the disease arereadily measurable by routine procedures familiar to a physician.

These antibodies may also be administered via genetic vectors thatexpress the paired heavy and light chains of a given antibody. This caninvolve a plasmid the efficiently expresses these genes or a viralvector, such as Adenoviral or Adeno-associated virus (AAV) vectors.These vectors can be delivered by injection into muscle tissue, and,depending on the dose, can secrete relatively large amount of secretedantibody into the circulation over a relatively long period of time.

Other therapeutic regimens may be combined with the administration ofthe anti-LAM and/or anti-PIM6/LAM antibodies of the present invention,for example, with another anti-LAM antibody, including but not limitedto those anti-LAM antibodies known in the art, e.g. murine anti-LAMantibodies or humanized versions thereof, or with a pharmaceuticalcompound, such as, but not limited to, antibiotics. Antibiotics that aresuitable for co-administration with the anti-LAM and/or anti-PIM6/LAMantibodies of the present invention include, but are not limited to,isoniazid, rifampin, rifapentine, ethambutol, pyrazinamide, bedaquiline,capreomycin, cycloserine, dexamethasone, kanamycin, and tinocordin. Thecombined administration includes co-administration, using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein preferably there is a timeperiod while both (or all) active agents simultaneously exert theirbiological activities. Such combined therapy can result in a synergistictherapeutic effect. The above parameters for assessing successfultreatment and improvement in the disease are readily measurable byroutine procedures familiar to a physician.

According to another embodiment, the present invention provides apassive vaccine or pharmaceutical compositions including at least oneanti-LAM and/or anti-PIM6/LAM antibody of the invention and apharmaceutically acceptable carrier. According to one embodiment, thevaccine or pharmaceutical compositions is a composition including atleast one antibody described herein and a pharmaceutically acceptablecarrier. The vaccine can include a plurality of the antibodies havingthe characteristics described herein in any combination and can furtherinclude other anti-LAM antibodies, including those of the presentinvention and those known in the art, e.g. murine anti-LAM antibodies orhumanized versions thereof. The passive vaccine may include one or morepharmaceutically acceptable preservatives, carriers, and/or excipients,which are known in the art.

According to another embodiment, the present invention covers an activevaccine or pharmaceutical composition including administering to patientat least one antigenic LAM or PIM6 epitope. The particular epitope to beemployed can be determined by testing the therapeutic activity ofantibodies described in this patent in an appropriate animal model forTB infection and/or pathogenesis. This model species can be mouse, orguinea pig, or rabbit, or primate. For example, if A194-01 is mostprotective then a vaccine bearing a form of the A194-01 epitope would beused, whereas if P30B9 is most protective, di-mannose substituted Ara6residues may be most effective at generating an appropriate humoralresponse. The active vaccine may include one or more adjuvants, whichare known in the art, e.g. alum, aluminum hydroxide, aluminum phosphate,paraffin oil, and cytokines, e.g. IL-1, IL-2, IL-12. The active vaccinemay comprise one or more pharmaceutically acceptable preservatives,carriers, and/or excipients, which are known in the art.

In some embodiments, the invention is directed to a recombinant vector,e.g. a plasmid, including a nucleic acid coding for an immunoglobulinheavy chain (Ig VH) of an anti-LAM antibody or an anti-PIM6/LAMantibody, and a second nucleic acid coding for an immunoglobulin lightchain (Ig VL). In other embodiments, the first nucleic acid and thesecond nucleic acid are in two different recombinant vectors. Accordingto another embodiment, the present invention covers a method of treatinga tuberculosis infection in an individual including administering tosaid individual a first nucleic acid coding for an immunoglobulin heavychain (Ig VH) of an anti-LAM or anti-PIM6/LAM antibody and a secondnucleic acid coding for an immunoglobulin light chain (Ig VL) of ananti-LAM or anti-PIM6/LAM antibody wherein each of the nucleic acids isoperably linked to a promoter region. The first nucleic acid and thesecond nucleic acid may be in a same recombinant vector or in twodifferent recombinant vectors. The recombinant vector may benon-replicating viral vectors, e.g. adeno-associated viruses (AAV), ormay be plasmids. In certain embodiments, the invention is directed to acell transformed with one or more vectors disclosed herein.

The above described antibodies and antibody compositions, vaccinecompositions, and vectors can be administered for the prophylactic andtherapeutic treatment of infection by virulent strains of theMycobacterium tuberculosis-complex.

H. Equivalents

Where a value of ranges is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges which may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference in their entireties.

As used herein and in the appended claims, the singular forms “a”, “and”and “the” include plural references unless the context clearly dictatesotherwise

The term “about” refers to a range of values which would not beconsidered by a person of ordinary skill in the art as substantiallydifferent from the baseline values. For example, the term “about” mayrefer to a value that is within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%,3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value, as well asvalues intervening such stated values.

Publications disclosed herein are provided solely for their disclosureprior to the filing date of the present invention. Nothing herein is tobe construed as an admission that the present invention is not entitledto antedate such publication by virtue of prior invention. Further, thedates of publication provided may be different from the actualpublication dates which may need to be independently confirmed.

Each of the applications and patents cited in this text, as well as eachdocument or reference, patient or non-patient literature, cited in eachof the applications and patents (including during the prosecution ofeach issued patent; “application cited documents”), and each of the PCTand foreign applications or patents corresponding to and/or claimingpriority from any of these applications and patents, and each of thedocuments cited or referenced in each of the application citeddocuments, are hereby expressly incorporated herein by reference intheir entirety. More generally, documents or references are cited inthis text, either in a Reference List before the claims; or in the textitself; and, each of these documents or references (“herein-citedreferences”), as well as each document or reference cited in each of theherein-cited references (including any manufacturer's specifications,instructions, etc.), is hereby expressly incorporated herein byreference.

The following non-limiting examples serve to further illustrate thepresent invention.

EXAMPLES

Example 1—the Methods Described Herein were Utilized to Culture Memory BCells in vitro and to molecularly clone immunoglobulin variable regiongenes to isolate several novel human monoclonal antibodies (mAbs)specific for LAM. One having ordinary skill in the art will recognizethat these methods described herein can be adjusted to selectivelyidentify rare antibodies with very high affinity, which could be presentas few as 1 out of 100,000 memory B cells circulating in the blood ofthe patient.

Monoclonal Antibodies

Murine monoclonal antibodies: Hybridoma cell lines producingLAM-specific murine monoclonal antibodies CS-35 and CS-40 obtained fromDr. Delphi Chatterjee's lab were recloned to homogeneity, and theantibodies were purified by protein A chromatography.

Antibodies 906.41, 906.7, 908.1 and 922.5 were provided by Dr. JohnSpencer, and FIND25 and FIND170 were provided by Tobias Broger at FIND.

Antigens

Mycobacterium tuberculosis derived H37Rv lipoarabinomannan (LAM)(NR-14848) and Mycobacterium smegmatis derived LAM (NR-14860) wereobtained from Colorado State University through BEI resources.LAM-derived glycoconjugates were synthesized in the Lowary lab.

ELISA Assays

Man-LAM (H37Rv) and PI-LAM (derived from Mycobacterium smegmatic) werediluted in CBC buffer (7.5 mM sodium carbonate, 17.4 mM sodiumbicarbonate, pH 9.0) and plated at a concentration of 100 ng/well in 96wells ELISA plates. After overnight incubation of plates at 4° C., wellswere washed with PBS, pH 7.4 containing 0.05% Tween-20 (PBST), thenblocked with 1% BSA (Sigma) in PBS buffer. PBST-washed plates wereincubated for 1 hour at 37° C. with plasma derived from individualsinfected with Mycobacterium tuberculosis and control plasma diluted inRPMI medium containing 2% FBS. PBST washed plates were then incubatedfor 1 hour with a 1:1000 dilution of alkaline phosphatase conjugatedgoat-anti-human IgG (γ-specific) (Millipore), or IgM (μ-specific)(Millipore), or IgA (α-specific). After PBST washing the color wasdeveloped with 50 μL of DEA buffer. The OD was measured at 405 nm byspectrophotometer. Titers were defined as the reciprocal dilution whichproduced an OD after subtracting the background OD taken the BSA coatedplate, and were determined by exponential interpolation.

Plasma Titrations

The two purified antigens were obtained from the BEI Repository, andwere plated overnight at 4° C. on 96 well ELISA plates at aconcentration of 2 μg/ml, and the plates were then blocked with 1% BSAin 1×PBS. The LAM-specific titers of plasma were tested by incubatingserially diluted samples at 37° C. for 1 hour, followed which the plateswere washed three times with PBS+0.1% Tween20. Bound antibody wasdetected with a mixture of alkaline phosphatase conjugated goatanti-human kappa and goat anti-human lambda at 1:1,000 dilution in 1%BSA in PBS and the signals were developed by adding alkaline phosphatesubstrate in DEA buffer. Reactivity was measured as OD405 at 30 min.

Human Subjects

Patients with active infection with Mycobacterium tuberculosis wereenrolled in the Lattimore practice at the Global Tuberculosis Institute.Active infection was defined by culture-proven tuberculosis disease or adiagnosis of clinical tuberculosis. This group included patients with arecent tuberculosis diagnosis, patients who were in the second month ofthe therapy. Uninfected patients were HIV-seronegative, tuberculin skintest-negative, healthy volunteers with no history of BacillusCalmette-Guerin (BCG) vaccination and negative for interferon-gammarelease assay (IGRA) (Quantiferon Gold In-Tube, Cellestis Inc, Valencia,Calif.). Informed written consent was obtained from participants, andthe study was approved by the Rutgers University Institutional ReviewBoard.

TABLE 4 Clinical characteristics of TB patients used in this studyDemographics of human subjects Sample ID Bleed date Treatment start dateDiagnosis Level of disease TB194 Mar. 3, 2014 Jan. 15, 2014 TST (+), AFBSmear (−), NAAT (+) Pulmonary TB TB210 Apr. 2, 2014 Mar. 12, 2014 TST(+), AFB Smear (+), Abnormal X-Ray Pulmonary TB TB256 Jun. 30, 2014 May19, 2014 TST (+), TBD (+), Abnormal X-Ray Pulmonary TB TB260 Jun. 10,2014 Jul. 3, 2014 TBD (+), AFB Smear (+), Abnormal X-Ray Pulmonary TBHC261 Jul. 3, 2014 NA LTBI (−), TST (−) LTBI (−), Non-contact TB310 Nov.13, 2014 Oct. 11, 2014 TBD (−), AFB Smear (−), IGRA (+) Pulmonary TBTB314 Nov. 18, 2014 Oct. 13, 2014 TBD (−), AFB Smear (−), IGRA (+)Pulmonary TB TB320 Dec. 1, 2014 Oct. 31, 2014 TBD (+), TST (+), AbnormalX-Ray Pulmonary TB TB366 Apr. 10, 2015 Mar. 17, 2015 TBD (+), TST (+),Abnormal X-Ray Pulmonary TB TB372 Apr. 15, 2015 Feb. 27, 2015 TBD (+),TST (+), Abnormal X-Ray Pulmonary TB TB373 Apr. 22, 2015 Mar. 3, 2015TBD (+), TST (+), Abnormal X-Ray Pulmonary TB TB384 May 5, 2015 Mar. 6,2015 TBD (+), TST (+), Abnormal X-Ray Pulmonary TB

1. Culture and Isolation of A194-01 (IgG Isotype)

Human monoclonal anti-LAM antibody A194-01, isotype IgG, was isolatedfrom cultured memory B cells obtained from a TB-infected patient,TB-194. A critical component of the in vitro culture system is thepresence of suitable feeder cells that can provide stimulation by CD40L,the ligand for CD40, a member of the TNF-receptor superfamily that isexpressed on the surface of B cells and plays an essential role inmediating T cell-dependent immunoglobulin class switching and memory Bcell development. Memory B cells were seeded on a feeder layer ofCD40L-expressing MS40L-low cells. These cells express a low level ofCD40L, and have been previously shown to efficiently support thereplication of memory B cells and their maturation to plasma cells (Luo,X., et al., Blood, 2009. 113(7). These cells were generated by infectingmurine stromal MS5 cells, that provide the B-lineage growth factor IL-7,with FUW-CD40L, a virus that transduces human CD40L, originally obtainedfrom Origene (Rockville, Md.). Memory B cells were isolated with theMACS human memory B cell isolation kit from Miltenyi (Cat.#130-093-546). Non-B cells were excluded from PBMCs by negativeselection with magnetic beads containing antibodies against the cellsurface marker CD2, CD3, CD14, CD16, CD36, CD43, CD56, CD66b andglycophorin A. To further eliminate naïve B cells, memory B cellsubpopulations were positively selected with magnetic beads coupled toantibody against the cell surface marker CD27, a marker for memory Bcells that is also expressed in low levels on plasma cells, but not onnaïve B cells. In the presence of CD40L-expressing feeder cells, theseconditions support the replication of the memory B cells and theirdifferentiation into plasmablasts secreting relatively high titers ofIgs into culture supernatants.

Cultures were refed at weekly intervals by replacing half of the culturesupernatant with fresh media. After 2-3 weeks there were sufficient Bcells to produce ˜1-5 μg/ml of secreted antibody. Assuming the presenceof 100-1,000 distinct clones in each well, this corresponded to anaverage concentration of 1-10 ng/mL of Ig per B cell clone. Thisconcentration is fairly low, and therefore this method was biasedtowards antibodies with relatively high affinities for the targetantigens. Approximately 80,000 cells memory B cells were purified fromthe blood of this patient and cultured in 96 wells of a 96 well cultureplate, for an initial density of ˜800 cells/well.

Culture supernatants were screened by ELISA for the presence ofantibodies against Mycobacterium tuberculosis-derived LAM. LAM wascoated at a concentration of 2 μg/mL in 50 μL of bicarbonate coatingbuffer per well of a 96 wells ELISA plate and incubated at 4° C.overnight. The plate was washed with PBST (0.1% Tween 20 in 1×PBS) 4times, and blocked with 200 μL of 2% nonfat milk in 1×PBS for 1 hour at37° C. 100 μl of the culture supernatant was added to the correspondingwells of the ELISA plate containing LAM, and incubated for 1 hour at 37°C. After additional washing steps, AP-conjugated mouse anti-humanFab-antibody was added to detect bound human antibody. After half anhour of incubation at 37° C., 100 μL of AP-substrate in DEA buffer wasadded to the ELISA wells and reactivity was determined colorimetricallyby measuring absorbance at 405 nm.

A positive signal (OD of ˜1 at 1 hr) was detected in only 1 well out of96 wells, indicating the rarity of these cells in this sample. Cellsfrom the positive cells were re-cultured at a density of 5-10 cells/wellin 10 wells of 96 well plates and rescreened for activity against LAM.This resulted in ˜ 6 positive wells (OD of ˜1 at 1 hr), again consistentwith the low frequency of LAM-reactive cells and suggesting that theoriginal positive well contained only a single LAM-positive B cellclone. Cells from several of the positive sub-clones were lysed and usedto isolate the variable regions of the H and L chains, which were thencloned into H and L chain expression vectors. A total of 10 diverse VHand 9 VL sequences were isolated from these wells, and these were thentested for activity by transfecting individual combinations in 293cells. Of 90 combinations tested, only a single combination of heavychain (p9045-IgG1-VH) and light chain (p9044-Vk) gave a positive signalagainst LAM. Antibodies were expressed by cotransfection ofcorresponding heavy and light chain plasmids in Expi-292 cells asdescribed by the manufacturer and grown in serum-free media. Antibodieswere purified by affinity chromatography on either protein A beads (forIgG) or protein L beads (for IgG), and eluted with low pH buffer. Thepurified antibodies were concentrated and characterized by SS-PAGE forsize and purity.

2. Isolation and Culturing of the IgM Isotype of P30B9

Human monoclonal anti-LAM antibody P30B9, isotype IgM, was isolated fromcultured memory B cells obtained from a TB-infected patient, TB-314.PBMCs were isolated from the blood of patient TB-314 by centrifugationon a ficoll gradient, and ˜30,000 memory B cells were purified asdescribed above with the MACS human memory B cell isolation kit fromMiltenyi. The purified memory B cells were cultured for 14 days byplating at 400 cells/well on monolayers of MS40-L cells grown in 96-wellplates, in the presence of IL-21 (100 ng/mL), IL-10 (100 ng/mL), IL-2(10 ng/mL), IL-4 (2 ng/mL), and CpG (1 μM), and cell supernatantsscreened by ELISA for binding to IH337Rv ManLAM. ManLAM was coated at aconcentration of 2 μg/mL in 50 μL of bicarbonate coating buffer per wellof a 96 wells ELISA plate and incubated at 4° C. overnight. The platewas washed with PBST (0.1% Tween 20 in 1×PBS) 4 times, and blocked with100 μL of 1% BSA in 1×PBS for 1 hour at 37° C. 50 μL of the culturesupernatant or diluted antibody were added to the corresponding wells ofthe ELISA plate containing LAM, and incubated for 1 hour at 37° C. Afteradditional washing steps, AP-conjugated goat anti-human IgG(H+L)-antibody was added to detect bound human antibody. After half anhour of incubation at 37° C., 50 μl of AP-substrate in DEA buffer wasadded to the ELISA wells and reactivity was determined by measuringyellow color at 405 nm. Only 1 out of 78 wells gave a positive signalwhen probed with a secondary goat anti-human IgG, IgA, IgM, kappa chainreagent. After expansion this well was transduced for BCL6 and Bcl-xL,linked by the self-cleaving porcine teschovirus-1 (P2A) peptide sequenceand followed by a GFP reporter gene is driven by IRES. These two genesstabilize memory B cells for long-term replication, and allow the cellsto be cultured even after selection of antigen positive cells byengagement of the BCR. The retroviral vectors were pseudotyped withGibbon ape leukemia virus (GaLV) envelope glycoprotein with the Rpeptide deleted from the C-terminal™ domain. Successful transduction ofprimary B cells led to expression of BCL-6, Bcl-xL and the markerprotein GFP. Viral titers were determined by counting GFP positive 293Tcells under the fluorescent microscope. Activated B cells weretransduced with retroviral vector in the presence ofpolybrene/retronectin.

After further expansion, the transduced cells were subcultured atlimiting dilutions in the presence of IL-21 (100 ng/mL) and IL-2 (10ng/mL). Well B9 on plate 30 (P30B9) was selected based on its strongLAM-binding activity and microscopic demonstration of the presence of asingle clone. The P30B9 supernatant bound exclusively to wells coatedwith H37Rv-LAM, and not with wells coated with LAM derived fromMycobacterium smegmatic or alpha crystallin. The cells from this wellwere lysed and RNA isolated using the RNeasy mini kit (Qiagen) followedby cDNA synthesis with oligo (dT), using the superscript III cDNAsynthesis system (Invitrogen). Antibody heavy and light chain variableregions were amplified by using Smith-Tiller's primers, and cloned inhuman heavy and light chain expression vectors. The heavy chain variableregion was initially cloned into a standard IgG vector. However, whencombined with the light chain sequence cloned into a human kappa chainexpression vector no LAM-binding activity was detected. At that point,the ManLAM-reactive antibodies produced in the original stablytransduced polyclonal well were re-probed with isotype-specificreagents, and found to be exclusively IgM. The P30B9 VH sequence wassubsequently cloned into an IgM H chain constant region expressionvector, and good binding activity was obtained upon co-transfection withthe corresponding kappa chain.

3. Characterization of Epitope Specificity of A194-01 IgG and P30B9 IgMand Murine Anti-LAM Antibodies Against LAM

A. To define the epitopes recognized by A194-01 IgG and P30B9 IgM, thebinding activities of said antibodies were compared to those of a numberof murine LAM-specific monoclonal antibodies (CS-35, CS-40, FIND25,FIND170, and the 900 series of monoclonal antibodies represented by908.1) against a series of 25 glyconjugates in which synthetic glycansrepresenting different structures present in LAM were conjugated tobovine serum albumin (FIG. 4A). These ranged in size from 4 to 26carbohydrate rings and represented the range of structural motifs knownto be present in various mycobacterial LAMs, including a number ofpoly-arabinose structures both uncapped and capped with phosphoinositol,alpha(1→2)-linked mono, di- and tri-Manp mannose structures, and5-deoxy-5-methylthiopentofuranosyl (MTX) motifs and various capped Ara4and Ara6 structures.

Six distinct reactivity patterns were obtained with this antigenic panelfor these monoclonal antibodies (FIG. 4B). The relative affinities ofthe monoclonal antibodies for these antigens were indicated by thetitration profile; high affinity reactions retain high reactivity at theintermediate dilution, whereas low affinity is indicated by a rapid dropin reactivity. The broadest pattern was seen for mouse mAb CS-35, whichreacted with modest affinity with LAM derived from Mycobacteriumtuberculosis and LAM derived from Mycobacterium smegmatis, andrecognized both capped or uncapped structures containing the basic Ara4and Ara6 motifs, consistent with the known specificity of this mAb forthe β-D-Araf-(1→2)-α-D-Araf-(1→5)-α-D-Araf-(1→5)-α-D-Araf motif.

The human monoclonal anti-LAM antibody A194-01 IgG also recognized alarge fraction of these structures and in many cases possessed thestrongest affinity. A109-01 IgG bound strongly to all uncapped Ara4 andAra6 structures and to the phosphoinositol-capped Ara4 structure, andless strongly to a subset of the mannose-capped structures. A109-01 IgGbound well with mono-mannose capped structures, but very weakly with thedi- and tri-mannose structures, although reactivity with the latterstructures was enhanced when the MTX substitution was present. Four ofthe 900 series of mouse monoclonal antibodies (represented by 908.1)reacted with relatively weak affinity with all uncapped Ara4 and Ara6structures, but not with any of the capped structures. Two mousemonoclonal antibodies from FIND (FIND25, also referred to as KI25),bound strongly with all Ara6 structures, irrespective of the presence ofabsence of capping, but did not recognize any Ara4 structures. CS-40,known to react specifically with ManLAM, reacted weakly with LAM derivedfrom Mycobacterium tuberculosis, and bound preferentially withmono-mannose-capped Ara4 and Ara6 structures.

The human monoclonal anti-LAM antibody P30B9 IgM, reacted strongly andwith high specificity with ManLAM derived from Mycobacteriumtuberculosis and with dimannose-capped Ara4 and Ara6 structures, andwith considerably weaker activity to the other mannose-containingstructures. Visualization of this residual activity is dependent on theassay conditions, and shows up in some assay formats (e.g., FIGS. 4 b ,8) but not in others (e.g., FIGS. 16, 18 ). Without wishing to be boundby theory, the relative specificity of P30B9 IgM for di-mannose cappedstructures is potentially clinically relevant, since terminal mannosylunits are known to mediate binding of lipoarabinomannan from virulentstrains of the Mycobacterium tuberculosis-complex to human macrophages,and, furthermore, di-mannose caps are known to be the dominantmodification of LAM derived from Mycobacterium tuberculosis.

Similar results were obtained when the epitope specificity of theA104-01 IgG and the P30B9 IgM were further mapped in a microarray assayagainst a larger panel of carbohydrate antigens. This panel includedseveral additional polymannose structures which were recognized by theP30B9 IgM, but not by any of the other antibodies tested (FIG. 8 ). Thiswas consistent with the P30B9 IgM preference for di-mannose capped Ara4and Ara6 structures, particularly, but not necessarily, those containingMan-α(1→2)-Man-α(1→5) linked to the terminal arabinose. The P30B9 IgMalso reacted strongly with a penta-mannose structure (59. AS-3-71) thatcontained the Man-α(1→2)-Man-α(1→6) but only weakly to a similarstructure containing the Man-α(1→3)-Man-α(1→6) (50. YB-BSA-18). Despiteits preference for the α(1→2) linkage, the P30B9 IgM did not react withAS-2-91, a tetra-mannose structure that contained the Man-α(1→2) linkagealong with an additional mannose linked α(1→6) to the second mannose.Without wishing to be bound by theory, this suggests that thespecificity of the IgM isotype of P30B9 may require that both sugars ofthe dimannose motif not contain any additional substitutions.

B. A more precise titration to map the fine specificities of thesemonoclonal antibodies towards the LAM-derived glycans demonstrated thecritical role of the terminal β-D-Araf-(1→2)-α-D-Araf-(1→5) disaccharidein antibody recognition of Ara4 structures. The Ara4 structure consistsof a β-D-Araf-(1→2)-α-D-Araf-(1→5)-α-D-Araf-(1→5)-α-D-Araftetrasaccharide, while the Ara6 structure contains an additionalβ-D-Araf-(1→2)-α-D-Araf-(1→3) disaccharide branch at the second sugar.Three of the monoclonal antibodies bound to both Ara4 and Ara6structures independent of mannose capping. All three monoclonalantibodies bound to the Ara4 structure (YB-8-099) and to YB-BSA-03,corresponding to the Ara4 structure with four additional α-D-Araf-(1→5)sugars at the reducing end (FIG. 9A). However, none of the monoclonalantibodies bound to a related octasaccharide (MJ-LZ-2) that contained aterminal β-D-Araf-(1→2)-α-D-Araf-(1→3) disaccharide, corresponding tothe lower branch of the Ara6 structure. This indicated that the upperbranch of the Ara6 structure containing theβ-D-Araf-(1→2)-α-D-Araf-(1→5) linkage was recognized by these monoclonalantibodies, and not the lower branch that contained theβ-D-Araf-(1→2)-α-D-Araf-(1→3) disaccharide.

The role of the terminal β-D-Araf-(1-2) linkage in antibody recognitionwas examined by probing the reactivity of these monoclonal antibodiesand the Ara6-dependent FIND25 antibody to three related polyα-D-Araf-(145) structures that contained truncated forms of the terminaldisaccharide (FIG. 9B). All three structures also contained an internalα-D-Araf-(143) branch. YB-BSA-07 terminated in a linear α-D-Araf-(145)structure, and was completely unreactive with all of the anti-LAMantibodies. YB-BSA-09 contained additional α-D-Araf sugars attached viaa (143) branch at the penultimate sugars of the two longer branches,resembling the structure of the Ara6 branch. This structure wasrecognized only weakly by the higher concentrations tested of the IgGisotype of A194-01 and by CS-35. YB-BSA-10 included terminalβ-D-Araf-(1-2) sugars at each of the branches, forming two complete Ara6structures at the non-reducing ends of the polysaccharide. Thisstructure was recognized by all of the monoclonal antibodies, withrelative binding strengths consistent with their affinities towards thenatural LAM antigen. These assays indicated that a terminalj-D-Araf-(1→2)-α-D-Araf-(1→5) disaccharide was a critical component ofall of the available Arabinose-reactive LAM-specific monoclonalantibodies.

C. A critical distinction between pathogenic strains of theMycobacterium tuberculosis-complex such as Mycobacterium tuberculosisand Mycobacterium bovis and non-pathogenic rapidly growing strains suchas Mycobacterium smegmatis is the presence of mannose-capped termini onthe pathogenic strains. As such, monoclonal antibodies that are specificfor distinct mannosylated structures could be useful for structuralstudies and for determining the functional contributions of thesemodifications. The activities of two of the monoclonal antibodiescharacterized in this study, CS-35 and FIND25/170, were completelyunaffected by the presence or absence of mannose caps. Binding of the900 series of monoclonal antibodies on the other hand was completelyabrogated by mannosylation of any sort (FIG. 4 ).

CS-40 on the other hand, bound only weakly with the unmodified Ara4glycan (YB-8-099) but strongly with Ara4 (YB-8-101) and Ara6 (YB-8-149)structures that contained single mannose caps. This experiment used amodified CS-40 in which the mouse heavy chain domain was substitutedwith the human IgG1 constant sequence, since this resulted in moresensitive detection of binding compared to the natural mouse antibodyused in FIG. 4 . The weak reactivity of CS-40 with the uncappedarabinofuranose structure was reflected in its weak reactivity with M.smegmatis LAM, compared to M. tb LAM. Attachment of an α(1-4) linked MSXsugar to the terminal mannose (i.e., YB-8-141 and YB-8-149) had noeffect on binding affinity, whereas attachment of a second α(1→2) linkedmannose sugar (YB-8-111 and YB-8-125) to generate a dimannose capcompletely abrogated CS-40 reactivity (FIG. 16 ).

A194-01 possessed a more complex reactivity pattern. A194-01 boundstrongly with uncapped arabinofuranosyl side chains and withmono-mannose capped Ara4 (YB-8-101) and Ara6 (YB-8-123) structures, butthis mAb reacted only weakly with the dimannose-capped Ara4 (YB-8-111)and even more poorly with tri-mannose capped Ara4 (YB-8-113) and almostnot at all for dimannose-capped Ara6 (YB-8-125). As was seen for CS-40,MTX substitution to the monomannose structures (YB-8-141, YB-8-149) didnot inhibit binding of A104-01, and of particular interest, MSX additionsignificantly improved recognition of the dimannose- andtrimannose-capped Ara4 structures (YB-8-133, YB-8-143). Consistent withthe high selectivity of P30B9 for ManLAM, the mAb bound specificallywith dimannose-capped Ara4 (YB-111) and Ara6 (YB-8-125) structures. Incontrast to the benign or beneficial effects of MSX substitution onbinding of CS-40 and A194-01, this substitution resulted in the completeloss of reactivity of P30B9, as did addition of an additional mannose toform the trimannose capped structures. These results suggested that thedifferent monoclonal antibodies recognized different regions andstructural aspects of LAM structure, with some binding solely to thearabinofuranose side chains and others binding with different levels ofspecificity to the capping motifs.

The relative binding specificities and affinities of the Ara6-reactiveantibodies were compared for representative glycoconjugates (FIG. 11 )The overall patterns were consistent with those obtained for the naturalantigens, PILAM and ManLAM (FIG. 3 ) and in the preliminary titrationagainst the glycoconjugates (FIG. 4 ). The human A194-01 IgG possessedhigher relative affinity for all of the uncapped structures and for theMSX-substituted Ara6-monomannose structure (YB-8-149), reacted withequal affinity with the Ara6 structure with single mannose caps, but didnot recognize the structures with di-mannose or tri-mannose caps. FIND25bound with similar or slightly higher affinity than CS-35 to allstructures that bore the standard Ara6 structure, both in capped oruncapped forms, but did not bind to two structures (YB-BSA-06 andYB-BSA-08) in which one of the branches was extended at the non-reducingend away from the branching point. 908.1 bound with weaker affinity toall of the uncapped structures, including the latter two, but did notrecognize any of the mannose capped structures 4.

Competition Studies Involving Anti-LAM Monoclonal Antibodies a, Overview

The ability of individual antibodies to compete for binding ofbiotinylated probe mAbs to LAM was titered by ELISA. Typical competitioncurves are shown in FIG. 16 for four of the anti-LAM antibodies,A194-01, CS-35, FIND25 and P30B9. As expected, the biotinylatedantibodies were all competed by their excess amounts of their unlabeledversions. Murine anti-LAM antibody 908.6 competed poorly, if at all,against the other antibodies. This was due to some extent to the weakaffinity of this antibody, but also reflects the restriction of 908.6binding to uncapped structures, and suggests that capped structures werethe dominant targets in ManLAM recognized by CS-35 and FIND25.

In agreement with its broad reactivity, CS-35 competed fully for bindingof all of the probe antibodies, although its competition withbiotinylated A194-01 was less potent than A194-01 for itself, consistentwith a lower affinity of CS-35 for LAM. Whereas CS-35 competed fullyagainst biotinylated FIND25, FIND25 competed only partially againstlabeled CS-35 (˜74% maximum competition), and even less effectivelyagainst A194-01 (˜50%). Without wishing to be bound by theory, thisresult presumably reflects the presence of Ara4 structures that arerecognized by A194-01 and CS-35, but not by FIND25, which bindsexclusively to the Ara6 motif. The fact that FIND25 competed with themajority of CS-35 binding suggested that Ara6 structures were morecommon than Ara4 structures. Despite its high affinity, A194-01 competedonly against itself, but not against either CS-35 or FIND25, furthersuggesting that the targets in LAM recognized by the latter twoantibodies predominantly consisted of structures (e.g. di-mannose andtri-mannose-capped structures) that are not recognized by A194-01. Incontrast to this result, A194-01 did compete fully and efficiently forbinding of FIND25 to the un-mannosylated PILAM, consistent with the rolefor efficient mannose capping of Ara6 structures in the lack ofcompetition in ManLAM.

Competition studies using the antibody P30B9 further supported theconclusion that the great majority of Ara6 structures in ManLAM werecapped with di-mannose, and that the bulk of dimannose caps resided onAra6 structures. P30B9 competed with ˜₇₀% of binding of FIND25 and ˜80%of the binding of CS-35 to ManLAM, confirming that the majority of thestructures recognized by these mouse mAbs were also recognized by P30B9.P30B9 binding to ManLAM was competed efficiently by itself, and by bothCS-35 and FIND25. The level of competition of P30B9 by FIND25 was closeto 100%, indicating that essentially all of the dimannose-dependentP30B9 binding sites were located on Ara6 sites, and few on Ara4structures. As expected, A194-01 competed very poorly for binding ofP30B9 to ManLAM, and 908.7 did not compete at all, consistent with thepoor recognition of dimannose-capped structures by these antibodies. Theinability of the latter antibodies to compete efficiently for binding ofP30B9 confirmed that this effect required binding of the competing mAbto the same branch as the probe mAb, and that binding to heterologousepitopes located on an adjacent branch of the same molecule did not leadto effective competition.

B. Relative A194-01 IgG and P30B9 IgM Affinities by Competition Assays

Mapping the reactivity of individual monoclonal anti-LAM antibodies,including the IgG isotype of A194-01 and the IgM isotype of P30B9, tospecific glycan structures allowed for characterization of thedistribution of said specific glycan structures in LAM by antibodycompetition studies (FIG. 10 ). These competition assays assumed that inorder for one antibody to compete for binding of a second (biotinylated,in cases where they are from the same species) antibody, the twoepitopes must be in close proximity to each other in the nativemolecule, potentially, but not necessarily, on the same or neighboringarabinan branch. This model was supported by asymmetric competitionpatterns, where for example, biotinylated IgG A194-01, which binds toboth uncapped, mono-mannosylated and MSX-substituted Ara4 and Ara6structures, competed efficiently by itself and by engineered variantsand/or derivatives of A194-01, but only partially by murine monoclonalantibody FIND25, which binds only to Ara6 structures. On the other hand,murine monoclonal antibody CS-35, which binds to all Ara4 and Ara6structures, gives more complete competition, although less efficiently,presumably due to its relatively low affinity.

The results of these assays revealed some surprising and unexpectedproperties. For example, the IgM isotype of P30B9, which binds to alldi-mannose capped ManLAM structures, was competed strongly andcompletely by itself, CS-35 and FIND170. Without wishing to be bound bytheory, the efficient competition of P30B9 by murine monoclonal anti-LAMantibody FIND25 suggests that the di-mannose capped structures in nativeLAM are largely localized to the Ara6 structures recognized by the FINDantibodies, and not appreciably expressed on Ara4 structures. Thisheightens the importance of being able to target and specifically bindto di-mannose capped Ara6 residues, as di-mannose capping is believed tobe the dominant form of LAM found in virulent strains of theMycobacterium tuberculosis-complex. The highly efficient competition ofbiotinylated FIND25 by the engineered variant IgM isotype of A194-01 isfurther evidence for the increased recognition of mannosylatedstructures by the IgM isotype of A194-01.

C. Competition Studies of A194-01 IgG and P30B9 IgM and Murine Anti-LAMAntibodies to ManLAM and PILAM

Binding competition assays between different anti-LAM monoclonalantibodies were used to analyze the distribution of various structuralforms in LAM. The IgG isotype of A194-01 recognized both unmodified Ara4and Ara6 side chains or chains that contained a single mannose cap, butdid not bind to side chains with either dimannose or trimannose cappingmotifs. The two FIND murine antibodies reacted with all forms of Ara6,but not with any Ara4 structures. P30B9 IgM was relatively specific forAra4 and Ara6 structures that contained dimannose caps.

Consistent with the broadening in reactivity for the A194-01 constructswith increased valencies, these constructs exhibited an increasedpotency in antibody competition activity. When tested for ability tocompete for binding of biotinylated A194-01 IgG against ManLAM, thedecameric A194-01 IgM and tetrameric scFv-IgG variants competed moreefficiently that the A194-01 IgG isotype itself (FIG. 11 ), thussignifying an increased potential therapeutic and diagnostic utility,while monomeric Fab and scFv forms competed less effectively (FIG. 1 ).The dimeric scFv engineered variant and/or derivative of A194-01competed equally as well as the A194-01 IgG isotype.

When the epitope specificity of the engineered variants and/orderivatives of A194-01 were compared to that of the A194-01 IgG, it wasobserved that they possessed broader reactivity (FIG. 14 ). Whereas theIgG isotype did not bind appreciably to the di-mannose (YB-8-123,YB-8-125) and tri-mannose (YB-BSA-113, YB-BSA-13) substitutedstructures, the IgM recognized these structures, and the scFv-IgG formpossessed increased activity against some of these structures as well.Because di-mannose capping, and especially di-mannose capped Ara6, isthe dominant LAM motif in virulent Mycobacterium tuberculosis, thissuggests a potentially enhanced utility of these engineered forms ofA194-01 in therapeutic and diagnostic applications.

Unlabeled A194-01 IgG competed against binding of biotinylated A194-01IgG to LAM derived from either Mycobacterium tuberculosis (ManLAM) (FIG.12A) or Mycobacterium smegmatis (PILAM) (FIG. 12D), whereas murinemonoclonal antibodies FIND170 and P30B9 were not able to compete forA194-01 binding to either antigen (FIG. 12A, D). This was consistentwith the dominant recognition of Ara4 structures that were recognized bythe A194-01 IgG isotype but not by either FIND170, which is specific forAra6, or P30B9 IgM, which is dependent on dimannose capping residues.Similarly, A194-01 did not compete with binding of either biotinylatedFIND25 (FIG. 12B) or P30B9 IgM (FIG. 12C) to ManLAM, consistent with thedifferent epitope specificities for these antibodies. In contrast to theinability of A194-01 IgG to compete for binding of FIND25 to ManLAM,A194-01 IgG competed strongly with ˜90% of the binding of FIND25 toPILAM (FIG. 12E), consistent with the known absence of mannose-cappingin PILAM and with the high affinity of A194-01 for the uncapped Ara4 andAra6 structures.

In contrast to the inefficient competition by A194-01 IgG, P30B9 IgMcompeted with ˜80% binding of FIND25, and FIND170 competed almostcompletely with binding of P30B9 (FIG. 12B, C). This strongly suggestedthat the great majority of the Ara6 structures recognized by the FINDmurine antibodies possessed di-mannose caps, and therefore were alsorecognized by P30B9 IgM, and that the majority of the dimannose-cappedstructures recognized by P30B9 were present on Ara6 structures. Thissuggests that di-mannose capped Ara6 is the dominant immunological motifin LAM motif from virulent Mycobacterium tuberculosis.

D. Additional Competition Studies

Additional competition studies were undertaken to highlight the factthat LAM is a complex antigen of undefined heterogeneity. The definitionof the different epitope specificities of LAM-reactive monoclonalantibodies allowed the use of binding competition assays to examine thedistribution of the various epitopes in native LAMs. The ability ofvarious antibodies to compete for binding of biotinylated probemonoclonal antibodies to LAM and synthetic glycoconjugates was titeredby ELISA. Typical competition curves for three monoclonal anti-LAMantibodies, A194-01 IgG, CS-35, and FIND25, are shown in FIG. 13A. Thebiotinylated probe monoclonal antibodies were competed by theirunlabeled versions when present in large excess. The murine monoclonalantibody 908.6 competed poorly, if at all, against the other antibodies.This was due to some extent to the weak affinity of this antibody, butalso reflected the restriction of 908.6 binding to uncapped structures,and further suggested that mannose-capped structures were the dominanttargets in ManLAM recognized by CS-35 and FIND25. Consistent with itsbroad reactivity, CS-35 competed for binding of biotinylated A194-01IgG, although less efficiently than did A194-01 IgG itself, consistentwith the higher affinity of A104-01 IgG for LAM. CS-35 also competedfully against biotinylated FIND25, while FIND25 competed only partiallyagainst labeled CS-35 (˜74% maximum competition) and even lesseffectively against A194-01 IgG (˜50%). This result presumably reflectsthe presence of Ara4 structures that are recognized by A194-01 IgG andCS-35, but not by FIND25, which binds exclusively to structurescontaining the Ara6 backbone. Despite its overall high affinity to LAM,A194-01 IgG competed only against itself, but not against either CS-35or FIND25. This suggested the sites in LAM recognized by the murinemonoclonal antibodies were dominated by dimannose and trimannose-cappedstructures that were not recognized by A194-01 IgG.

Additional competition studies using P30B9 IgM, which binds specificallyto di-mannose capped ManLAM, further supported the clinicallysignificant conclusion that the great majority of Ara6 structures inManLAM were capped with di-mannose, and that the bulk of di-mannose capsresided on Ara6 structures in Mycobacterium tuberculosis derived ManLAM.P30B9 IgM competed with ˜80% of binding of FIND25 to ManLAM (FIG. 13B),consistent with the majority of the Ara6 structures recognized by FIND25also being recognized by P30B9 IgM. P30B9 IgM did not compete for FIND25binding to PILAM, consistent with the absence of the P30B9 dimannoseepitope in PILAAM, due to the lack of mannosylation in PILAM.Furthermore, the A194-01 IgG did not compete for binding of FIND25 toManLAM, again consistent with the great majority of the Ara6 structuresbearing di-mannose caps, which are not recognized by the A194-01 IgG.Confirming the role of mannosylation in this effect, A194-01 IgGcompeted very efficiently for binding of FIND25 to PILAM, consistentwith the high affinity of A194-01 for PILAM and the absence of mannosecapping in this antigen.

Competition data for the binding of biotinylated P30B9 IgM furthersupported this conclusion. Binding of biotinylated P30B9 IgM wascompeted most efficiently by itself and with equal efficiency by CS-35and FIND25, but only weakly and incompletely by A194-01 IgG (FIG. 13C).The level of competition by FIND25 was close to 100%, indicating thatessentially all of the di-mannose-dependent P30B9 IgM binding sites werelocated on Ara6 structures. Consistent with this interpretation, CS-35also competed for binding of P30B9 to dimannose-capped Ara4 (YB-8-111)and dimannose-capped Ara6 (YB-8-125), whereas FIND170 competed only forthe latter antigen and the A194-01 IgG competed for neither. The generalsimilarity between the competition curves for ManLAM and the homogeneousYB-8-125 glycoconjugates indicated that the competition resultscorrelated with the presence or absence of the relevant epitopes on asingle carbohydrate side-chain, and that indirect steric effects due tobinding of antibodies to more distant heterologous sites played littleif any role in competition.

One surprising and unexpected result is the complete lack of competitionbetween A194-01 IgG versus CS-35 and FIND25. The ability of CS-35 tocompete for binding of A194-01 IgG to ManLAM was expected due to therecognition of all A194-01 IgG targets by CS-35, and the less efficientcompetition by CS-35 than by A194-01 IgG itself is consistent with therelative affinities of these antibodies for ManLAM (FIG. 3 ). Similarly,the incomplete competition of A194-01 IgG binding by FIND25 can beexplained by the presence of Ara4 targets recognized by the former butnot the latter antibody.

The efficient and complete competition of binding of biotinylated P30B9IgM by both CS-35 and FIND170 suggests that the di-mannose capsrecognized by P30B9 IgM were present almost exclusively of Ara6structures recognized by the FIND mAb, which is of clinical anddiagnostic significance. This was supported by the relatively efficientcompetition of binding of FIND25 by P30B9 IgM, which blocked ˜80% of thebinding activity of FIND25 to ManLAM but had no effect for PILAM. Thisstrongly suggests that ˜80% of the Ara6 sites in ManLAM recognized byFIND25 contained di-mannose caps, and that essentially all of thedi-mannose caps are present on Ara6, and not on Ara4 structures. Takentogether with the inability of A194-01 IgG to compete with CS-35 orFIND25, these results demonstrate that di-mannose-substituted Ara6 wasthe dominant immunogenic structure on ManLAM derived from Mycobacteriumtuberculosis, and thus represents a highly important antigenic target.

Studies of the LAM-specific antibody responses in patient plasmaindicate that the response is dominated by IgG2 isotypes directedagainst linear Ara4/Ara6 structures, independent of mapping. Theefficient competition of P30B9 IgM by IgG monoclonal anti-LAM antibodiesspecific for arabinofuranose-dependent epitopes such as CS-35 andFIND25, suggests that the dominant IgG2 responses against such epitopesin patient plasma would also compete for di-mannose-dependent anyManLAM-specific antibodies that may be produced in lower titers. Thus,even if the latter class of antibodies might have more effectiveanti-bacterial activities, it is likely that these effects may belimited by the competition for binding by the dominant, non-functionalIgG2 antibodies directed against arabinose-dependent epitopes that arepresent in patient sera. Without wishing to be bound by theory, thedominant humoral response may actually protect the bacteria againstpotential effects of rarer antibodies, such as multivalent antibodieslike P30B9 IgM, or the engineered variants and/or derivatives ofA194-01, such as the pentavalent IgM isotype or the tetravalent scFv-IgGthat could provide immune control against infection and pathogenicity.

5. Effects of Valency on A194-01 Binding

The discovery that the reactivity of the dependence ofdi-mannose-reactive P30B9 on its IgM isotype suggested that multivalencycan contribute to the affinity of antibodies towards LAM. Withoutwishing to be bound by theory, this suggested that a single LAM moleculepossessed multiple antibody-binding sites or epitopes, consistent withthe known branched structure and complexity of LAM, and that antibodieswith higher valencies were able to bind to more sites that divalentantibodies, resulting in greater affinities. The effect of antibodyvalency towards binding efficiency to LAM was examined for variousengineered variants and/or derivative forms and/or isotypes of the humanmonoclonal antibody A194-01 in a binding competition assay, usingbiotinylated A194-01 IgG as the target. The antibody forms included amonovalent single chain scFv in which the VH and VL regions are joinedby a flexible peptide linker, a monovalent Fab protein, a dimeric scFvprotein in which two scFv domains were joined by a flexible linker, thenatural dimeric IgG (FIG. 1A), and two higher valent forms, atetravalent A194-01 scFv-IgG, and a pentavalent (decavalent for bindingsites) IgM isotype (FIG. 1B). Converting the intact divalent IgG to amonovalent Fab resulted in a very large loss of binding activity, witha >100-fold increase in concentration required to compete for 50% of thebinding activity of the biotinylated A194-01 IgG, compared to the IgGagainst itself (FIG. 1C). The single chain also competed inefficiently,with a 33-fold decrease in activity. On the other hand, the scFv dimercompeted with similar efficiency as the IgG isotype of A194-01. Withoutwishing to be bound by theory, this suggested that the efficient bindingof the divalent forms of A194-01 to LAM required the attachment of bothbinding sites to adjacent targets in a single molecule of the antigen.The higher valent forms competed more efficiently, on a molar basis.This may be simply due to the presence of additional binding sites, butmay also reflect an increased affinity of the higher valent forms.

The specificity of both the tetrameric scFv-IgG variant and/orderivative of A194-01 and the decameric IgM isotype of A194-01 werecompared to that of the IgG isotype of A194-01 against the syntheticglycoconjugate panel described above (FIG. 14 ). This revealed that bothengineered scFv-IgG variant and the engineered IgM isotype possessedbroader reactivities with some of the glycans that the IgG isotyperecognized only weakly or not at all. Enhanced binding was seen with thedi-mannose capped structures (YB-8-111, YB-8-125, YB-8-133) and withsome of the tri-mannose capped structures (YB-8-113, YB-8-143,YB-BSA-13) with the engineered IgM isotype of A194-01 possessing thebroadest reactivity panel. This has significant diagnostic andtherapeutic potential, given the significance of di-mannose capping.Interestingly, enhanced reactivity was also seen for the A194-01 IgMisotype with an arabinose structure that was missing the terminalβ-D-Araf-(1→2) linkage (YB-BSA-09), which was not recognized by any ofthe monoclonal anti-LAM antibodies tested. Without wishing to be boundby theory, this suggested that the increased valency resulted in astrengthening of the avidity of the IgM isotype of A194-01 for its basicarabinose-containing epitope, to the point that inhibitory effects ofterminal substitution were overcome.

The reactivity profiles of A194-01 IgG and the engineered IgM andscFv-IgG forms of A194-01 were further analyzed by reciprocal bindingcompetition experiments against either ManLAM or PILAM. Whereas allthree forms cross-competed for binding of the biotinylated antibodies toPILAM and for binding of A194-01 IgG to ManLAM, only the higher-valentscFv-IgG and IgM forms of A194-01 competed efficiently with binding ofthe modified antibodies to ManLAM. These differences in competitionactivities were consistent with a broader binding profile of theengineered forms, and suggested that they bound to sites on LAM thatwere not recognized by the IgG form.

This conclusion was further supported by reciprocal competition studiesin which the different isotypes of A194-01 competed with binding ofbiotinylated FIND25 IgG and P30B9 IgM to ManLAM. Whereas the A194-01 IgGdid not compete with binding of P30B9 IgM and FIND25 to ManLAM, both thescFv-IgG and IgM forms of A194-01 competed almost completely withbinding of these two monoclonal antibodies (FIG. 15A, B). As expected,all three forms of A194-01 competed with binding of FIND25 to PILAM(FIG. 15C) consistent with the absence of mannosylation in PILAM. Thisenhanced activity suggested that these modifications would enhance thepotential utility of this antibody for immunodiagnostic applications,and may also increase the effectiveness of these reagents asimmunotherapeutic purposes as well.

Example 2—Isolation and Characterization of Novel Human MonoclonalAntibodies Specific for Glycolipids of M.tb-Isolation of First mAbSpecific for an Epitope Shared by LAM and PIM6

P95C1 antibody heavy and light chain were isolated from a single B cellclone, by screening for reactivity with ManLAM, from a patient withlatent tuberculosis infection (LTBI). P95C1 is an IgM isotype antibody,which binds to both ManLAM and PILAM. This was shown by glycoconjugatebinding studies that indicated that P95C1 did not bind to any moleculesthat expressed various arabinose side-chains, either uncapped or cappedwith various mannose structures. The only structures recognized were twopolymannose structures that possessed structural motifs present in themannan base that were conserved between PIM6 and various LAMs (FIGS.18(A), 18(B), 18(C)). This LAM-PIM6 crossreactivity was confirmed by awestern blot assay showing that whereas A194-01 and P30B9 bound only toLAM, P95C1 also reacted with LAM precursor glycolipids molecules, LM andPIM6 (FIGS. 20(A), 20(B)).

Although P95C1, like P30B9, was naturally expressed as an IgM, incontrast to P30B9 it retained reactivity when converted to either theIgA or IgG isotype (FIG. 19 ). This may be a reflection of the nature ofthe nature of the epitope or its location in the mannan region of theLAM molecule, or may be related to the higher number of mutations in theP95C1 variable regions, consistent with a more mature antibody sequence.The variable regions of the P95C1 heavy and light chain have 19 and 13amino acid point mutations respectively from its closet germlineantibody sequence.

It has recently been shown that antibodies made by individuals withlatent disease are functionally superior from those with activetuberculosis in promoting phagolysosomal fusion, inflammasomeactivation, and macrophage killing of internalized mycobacteria (Lu etal. 2016). It is therefore of interest that P95C1 was isolated from aLTBI patient and it has more mutations in its variable region than P30B9and two other LAM-specific mAbs that were isolated from the same LTBIpatient that possess distinct ManLAM epitope specificity.

Little is known about the nature of the human humoral immune responseagainst M.tb infection. Although it is widely known that surfaceglycolipids of M.tb contribute to inhibition of the activity ofmacrophages and dendritic cells there is contradictory information aboutwhether mannose-capped lipoarabinomannan (ManLAM) orphosphatidylinositol mannoside 6 (PIM6) is the major immunoinhibitorysurface component of M.tb. This question is further complicated by thecommon contamination of purified preparations of PIM6 by ManLAM, andvice versa. One way of addressing this question is to test the abilityof antibodies specific for these two modulators to inhibit theseinhibitory activities. However, this has not been possible due to theabsence of well-characterized antibodies that are specific for these twoantigens. To date, there have been no antibodies reported that recognizePIM6, and this invention describes the first high affinity mAb thatrecognizes PIM6. The PIMs (PIM2 and PIM4) are precursors for ManLAM, andthere is some structural relationship between the mannan domain ofManLAM and the polymannose structure of PIM6.

Antibodies can exert their functions in two ways 1) by direct blockingof host cell invasion and neutralization of bacterial products 2)indirectly through Fc-mediated complement and cell activation mechanismsthrough Fc receptors. Antibody-mediated effector function are greatlyaffected by the antibody isotype. A recent study showed humanisotype-dependent inhibitory antibody responses against M.tb anddemonstrated that IgA, but not IgG, antibodies specific for differentM.tb surface antigens can block M.tb uptake by lung epithelial cellsindependent of the expression of IgA Fc receptors. To test the effect ofP95C1 isotypes on LAM binding, the constant region of P95C1-IgM heavychain was replaced by CH-IgA and CH-IgG to generate P95C1-IgA andP95C1-IgG. The binding affinity of P95C1 isotypes (IgM, IgA, IgG) withManLAM and PILAM were comparable (FIG. 19 ).

Other Embodiments

Any improvement may be made in part or all of the antibodies,compositions, kits and methods. All references, including publications,patent applications, and patents, cited herein are hereby incorporatedby reference. The use of any and all examples, or exemplary language(e.g., “such as”) provided herein, is intended to illuminate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. Any statement herein as to the nature orbenefits of the invention or of the preferred embodiments is notintended to be limiting, and the appended claims should not be deemed tobe limited by such statements. More generally, no language in thespecification should be construed as indicating any non-claimed elementas being essential to the practice of the invention. This inventionincludes all modifications and equivalents of the subject matter recitedin the claims appended hereto as permitted by applicable law. Moreover,any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contraindicated by context.

What is claimed:
 1. A monoclonal anti-lipoarabinomannan (anti-LAM)antibody, Or an antigen-binding portion thereof, that specifically bindsto a LAM epitope comprising an Ara4 structure, an Ara6 structure, or acombination thereof, wherein the anti-LAM antibody comprises a CDR1light chain variable region having at least 80% identity with SEQ ID NO:1 or antigenic fragments thereof, a CDR2 light chain variable regionhaving at least 80% identity with SEQ ID NO: 2 or antigenic fragmentsthereof, a CDR3 light chain variable region having at least 80% identitywith SEQ ID NO: 3 or SEQ ID NO: 26 or antigenic fragments thereof, aCDR1 heavy chain variable region having at least 80% identity with SEQID NO: 4 or antigenic fragments thereof, a CDR2 heavy chain variableregion having at least 80% identity with SEQ ID NO: 5 or antigenicfragments thereof, and a CDR3 heavy chain variable region having atleast 80% identity with SEQ ID NO: 6 or SEQ ID NO: 23 or antigenicfragments thereof, having a modified heavy chain constant region.
 2. Themonoclonal anti-LAM antibody or antigen-binding portion thereof of claim1, said antibody comprising a heavy chain variable region comprising theamino acid sequences of SEQ ID NO:21 and SEQ ID NO:23, and a light chainvariable region comprising the amino acid sequences of SEQ ID NO: 24 andSEQ ID NO:26.
 3. The monoclonal anti-LAM antibody or antigen-bindingportion thereof of claim 1, wherein the antibody is an scFv-IgG, an IgAor an IgM antibody.
 4. The monoclonal anti-LAM antibody or antigenbinding portion thereof of claim 1, wherein the antibody is a humanantibody, a humanized antibody, or a chimeric antibody.
 5. A method ofdiagnosing an active tuberculosis infection in an individual comprising:(a) obtaining a sample from an, individual that comprises or issuspected of comprising lipoarabinomannan (LAM); (b) contacting thesample with a first antibody that binds specifically to an epitopepresent on a LAM molecule; (c) contacting the sample with a detectionantibody that binds specifically to a different binding site in the LAMmolecule than the binding site bound by the first antibody; (d)detecting binding of the detection antibody to the different bindingsite in the LAM molecule; and (e) diagnosing the patient as having anactive tuberculosis infection, wherein capture of LAM by the firstantibody indicates an active tuberculosis infection, and wherein atleast one of the antibodies is a monoclonal anti-LAM antibody, or anantigen-binding portion thereof, that specifically binds to a LAMepitope comprising an Ara4 structure, an Ara6 structure, or acombination thereof, wherein the anti-LAM antibody comprises a CDR1light chain variable region having at least 80% identity with SEQ. IDNO: 1 or antigenic fragments thereof, a CDR2 light chain variable regionhaving at least 80% identity with SEQ ID NO: 2 or antigenic fragmentsthereof, a CDR3 light chain variable region having at least 80% identitywith SEQ ID NO: 3 or SEQ ID NO: 26 or antigenic fragments thereof, aCDR1 heavy chain variable region having at least 80% identity with SEQID NO: 4 or antigenic fragments thereof, a CDR2 heavy chain variableregion having at least 80% identity with SEQ ID NO: 5 or antigenicfragments thereof, and a CDR3 heavy chain variable region having atleast 80% identity with SEQ ID NO: 6 or SEQ ID NO: 23 or antigenicfragments thereof.
 6. The method of claim 5, wherein the detectionantibody is a monoclonal anti-LAM antibody, or an antigen-bindingportion thereof, that specifically binds to a LAM epitope comprising anAra4 structure, an Ara6 structure, or a combination thereof, wherein theanti-LAM antibody comprises a CDR1 light chain variable region having atleast 80% identity with SEQ ID NO: 1 or antigenic fragments thereof, aCDR2 light chain variable region having at least 80% identity with SEQID NO: 2 or antigenic fragments thereof, a CDR3 light chain variableregion having at least 80% identity with SEQ ID NO; 3 or SEQ ID NO: 26or antigenic fragments thereof, a CDR1 heavy chain variable regionhaving at least 80% identity with SEQ ID NO: 4 or antigenic fragmentsthereof, a CDR2 heavy chain variable region having at least 80% identitywith SEQ ID NO: 5 or antigenic fragments thereof, and a CDR3 heavy chainvariable region having at least 80% identity with SEQ ID NO: 6 or SEQ IDNO: 23 or antigenic fragments thereof.
 7. The method of claim 5, whereinthe detection antibody is an anti-LAM antibody that binds specificallyto LAM, and wherein the first antibody or the detection antibodycomprises a CDR1 light chain variable region having at least 80%identity with SEQ ID NO: 1 or antigenic fragments thereof, a CDR2 lightchain variable region having at least 80% identity with SEQ ID NO: 2 orantigenic fragments thereof, a CDR3 light chain variable region havingat least 80% identity with SEQ NO: 3 or SEQ ID NO: 26 or antigenicfragments thereof, a CDR1 heavy chain variable region having at least80% identity with SEQ ID NO: 4 or antigenic fragments thereof, a CDR2heavy chain variable region having at least 80% identity with SEQ ID NO:5 or antigenic fragments thereof, and a CDR3 heavy chain variable regionhaving at least 80% identity with SEQ ID NO: 6 or SEQ ID NO: 23 orantigenic fragments thereof.
 8. The method of claim 5, wherein thedetection antibody is an anti-LAM antibody that binds specifically toLAM, and wherein the first antibody or the detection antibody is anscFv-IgG or IgM antibody and comprises a CDR1 light chain variableregion having at least 80% identity with SEQ ID NO; 1 or antigenicfragments thereof, a CDR2 light chain variable region having at least80% identity with SEQ ID NO: 2 or antigenic fragments thereof, a CDR3light chain variable region having at least 80% identity with SEQ ID NO:3 or SEQ ID NO: 26 or antigenic fragments thereof, a CDR1 heavy chainvariable region having at least 80% identity with SEQ ID NO: 4 orantigenic fragments thereof, a CDR2 heavy chain variable region havingat least 80% identity with SEQ ID NO: 5 or antigenic fragments thereof,and a CDR3 heavy chain variable region having at least 80% identity withSEQ ID NO: 6 or SEQ ID NO: 23 or antigenic fragments thereof.
 9. Themethod of claim 5, wherein the individual is a human.